CN1061623A - The ciliary neurotrophic factor receptor - Google Patents

Abstract

The present invention relates to ciliary neurotrophic factor (CNTF) acceptor, and CNTF receptor nucleic acids and aminoacid sequence are provided.Also relate to (I) and measure the active detection system of CNTF; (II) experimental model system of research CNTF physiological role; (III) diagnostic techniques of the europathology state that evaluation CNTF is relevant; (IV) treatment technology of the N﹠M pathological state that treatment CNTF is relevant; And (V) identify with the method that comes from the molecule of CNTF and CNTFR.

Description

The ciliary neurotrophic factor receptor

1, foreword

The present invention relates to the ciliary neurotrophic factor receptor (CNTER), and CNTF acceptor coding nucleic acid and aminoacid sequence are provided.The present invention also relates to (ⅰ) for detecting the active detection system of CNTF; (ⅱ) be the test model system of the physiological role of research CNTF; (ⅲ) for identifying the diagnostic techniques of the neural state that CNTF is relevant; (ⅳ) for handling the treatment technology of the relevant neural state of CNTF; And (ⅴ) for differentiating the method with the CNTFR homolgous molecule.

2, background of invention

2.1. ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a kind of required protein (Manthorpe et al., 1980, J.Neurochem.34: 69-75) of the external survival of embryo chicken ciliary ganglion neurone that specifically be.On this ciliary ganglion anatomy in the side rectus of optic nerve and the socket of the eye chamber scope between the sheath; It holds the parasympathetic fibers from the oculomotor nerve of domination ciliary muscle and sphincter muscle of pupil.

Have been found that the ciliary ganglion neurone belongs to the neurone colony that presents definite necrocytosis cycle.In the chicken ciliary ganglion, the half neurone that has existed when having observed 8 days embryonic stages (E8) is dead (Landmesser and Pilar, 1974, J.Phy-siol.241: 737-749) before E14.In this identical time cycle, the ciliary ganglion neurone just with its target tissue, promptly Yan ciliary body and choroid form in succession.Landmesser and Pilar(1974, J.Physiol.241: 751-736) observe, before the necrocytosis cycle, remove a glance, cause the neuronic loss fully of ciliary ganglion in pleural ganglion.On the contrary, Narayanan and Narayanan(1978, J.Embryol.Ex.Morphol.44: 53-70) observe, thereby increase the amount of available target tissue, can reduce the death of ciliary ganglion neuronal cell by implanting an eye original hase that replenishes.These results are consistent with the existence that acts on the neuronic target deutero-of ciliary ganglion human BDNF.

When cultivating, find that ciliary ganglion (CG) neurone needs a factor or a plurality of factor in order to survive.Identified the activity of ciliary neurotrophic factor in the following cases: (Helfand et al., 1976, Dev.Biol.50: 541-547 in the chicken muscle cell conditioned medium; Helfand et al., 1978, Exp.Cell Res.113: 39-45; Bennett and Nurcome, 1979, Brain Res.173: 543-548; Nishi and Berg, 1979, Nature 277: 232-234; Varon et al., 1979, Brain Res.173: 29-45), (McLennan and Hendry, 1978, Neu-rosci.Lett.10: 269-273) in the muscle extract, (Varon et al., 1979, Brain Res.173: 29-45 in the Embryo Gallus domesticus extract; Tuttle et al., 1980, Brain Res.183: 161-180), and in the substratum of adjusting by heart cell (in order to discuss, also can consult Adler et al., 1979, Science 204: 1434-1436 and Barbin et al., 1984, J.Neurochem.43: 1468-1478).

(1979, Science 204: 1434-1436) used a kind of test macro based on the neuronic little well culture of CG to find profuse CNTF source with proof in the innerv intraocular target tissue of CG neurone for people such as Adler.In 8000 kilonems (TU) scope that existed among the embryo in 12 days, found that 2500TU is present in the ocular tissue; It seems that activity be confined to contain ciliary body and choroidal part.

Thereafter, and people such as Barbin (1984, J.Neurochem.43: 1468-1478) reported method by enrichment CNTF in the Embryo Gallus domesticus ocular tissue.Also find the active right and wrong CG tissue of CNTF, comprised (Williams et al., 1984, Int.J.Develop.Neurosci.218: 460-470) that rat sciatic nerve is relevant.People such as Manthrope (1986, Brain Res.367: 282-286) reported and use a kind of being similar to by the active used separation circuit of separation of C NTF in the corn by the Mammals CNTF activity of purifying of part in the rat sciatic nerve extract of growing up.In addition, Watters and Hendry(1987, J.Neurochem.49: 705-713) disclose a kind of use heparin-affinity chromatograph method under non-sex change condition by the OX-heart tissue in the about 20000 times method of the active enrichment of CNTF.CNTF activity (Manthorpe et al., 1983, Brain Res.267: 47-56 in the cerebral tissue that damages have also been identified; Nieto-Sampedro et al., 1983, J.Neurosci.3: 2219-2229).

People such as Carnow (1985, J.Neurosci.5: 1965-1971) and people such as Rudge (1987, Develop.Brain Res.32: 103-110) disclose by the Western trace of tissue extract and identify that class CNTF is active and identify and contain the method that the active protein belt of CNTF and use reactive dyestuffs identification of cell are survived and distinguished by in culture dish, cultivate the nitrocotton bar with the CG neurone subsequently.Use this method, people such as Carnow (1985, J.Neurosci.5: 1965-1971) observe the active and chicken CNTF(20.4kD of adult rat sciatic nerve and brain derived CNTF) compare and look and present different size (24kD).

Recently, CNTF is cloned in bacterial expression system and is synthesized, is 07/,570 651 as people such as Sendtner at application number, name is called " Ciliary Neurotrophic Factor ", the applying date is described in the U.S. Patent application in August 20 nineteen ninety, at this it is all added as a reference.Use the recombinant chou probe, the seemingly about 1.2kb of the size of the CNTF-mRNA in the adult rat tissue.It is coded by the open mRNA that reads yard of the weak point 5 with 77bp ' do not translate district and 600bp that rat brain CNTF is cloned and is found, indicated a kind of about 200 amino acid whose protein (Stockli et al., 1989, Nature 342: 920-923).Human body CNTF is also by clone and sequencing (people's such as Sendtner U.S. Patent application: application number is 07/570651, and name is called " Ci-liary Neurotrophic Factor ", and the applying date is August 20 nineteen ninety); Its encoding sequence is saved corresponding to the sequence of its rat basically, and encoding sequence is not preserved less.

2.2. the function character of ciliary neurotrophic factor

A series of physiological effects have been considered to because CNTF causes.As mentioned above, to be said to be at first be activity as the component-survival of neurone-a kind of parasympathetic nervous system of supporting E8 chicken ciliary ganglion to CNTF.Other biological property to the known CNTF of containing active ingredient is described below:

People such as Saadat (1989, J.Cell.Biol.108: 1807-1816) observe the preparation that the top of rat sciatic nerve CNTF purifies and induced the orthosympathetic cholinergic differentiation of rat in the culture.Also have Hoffman(1988, J.Neurochem.51: 109-113) found to have improved the level of choline-O-acetyltransferase activity in the retina unimolecular layer culture by the CNTF activity of corn derivative.

(1988, Nature 335: 70-73) studied the colony by bipotentiality colloid progenitor cell in term mouse optic nerve and the brain derived culture for people such as Hughes; Having demonstrated these progenitor cells at first makes the generation oligodendroglia and produces 2 type stellate cells subsequently.Under the culture condition that is adopted, it seems that the differentiation of oligodendroglia be that progenitor cell by an oligodendroglia 2 type stellate cells (0-2A) directly forms, and it seems that the Astrocytic differentiation of 2 types need exist a kind of induced protein that is similar to or is equal to CNTF (also can be referring to Anderson, 1989, Trends Neurosci.12: 83-85).

Heymanns and Unsicker(1979, Proc.Natl.Acad.Sci.U.S.A.4: 7758-7762) observe, the high speed supernatant liquor of the cell extract of neuroblastoma produces those the relevant effects of CNTF activity that are similar to from corn or rat sciatic nerve; Shown with CNTF similar but be not equal to the proteinic existence of (with regard to molecular weight).

Ebendal(1987, J.Neurosci.Res.17: 19-24) studied class CNTF activity in multiple rat and the chicken tissue.He observes the class CNTF activity in the rat of quite wide scope, but does not observe in the chicken tissue; Found that mouse liver, splenic t-cell and lower jaw glandular cell are relevant with low-level CG survivin promoter activity, and heart, brain and skeletal muscle tissue are relevant with higher survivin promoter activity.In the tissue of being tested, it is active relevant with the mouse kidney to observe the highest class CNTF.

Although above-mentioned research shows, many tissues and cell extract contain the activity (being that they are supported in the neuronic survival of E8 chicken ciliary ganglion in the tissue culture bioassay) that support also responds the neurone population survival of CNTF, but can not think that protein single or that be equal to is to respond that these are active.Shown in this family of fibroblast growth factor (FGFG) (Dionne et al., 1990, EMBO J.9: 2685-2692), for example, a series of different polypeptide or protein separately have identical physiologically active in the physiology calibrating.

The neuronal specificity of having found corn and rat sciatic nerve CNTF at first has some overlapping with the neurone colony that responds NGF.Has some eclipsed neuron behavior although observed CNTF and NGF, but CNTF and NGF's does the time spent in the neurone colony that research is grown, distinguishing characteristics between them become the most obviously (Skaper and Varon, 1986, Brain Res.389: 39-46).Except their not same-action when growing, also can be according to molecular weight, iso-electric point, can not support the ability of the external survival of CGF neurone CNTF and NGF to be distinguished (Barbin et al. by NGF antibody inactivation and according to CNTF, 1984, J.Neurochem.43: 1468-1478).(1989, Science 246: the sequence of 1023-1026) having reported the protein homology that CNTF do not report with any prior art for people such as Lin.(application number is 07/,570 651 to people such as Sendtner, name is called " Ciliary Neuro-trophic Factor ", the applying date is the U.S. Patent application in August 20 nineteen ninety) observe recombinant chou CNTF start in the survival of back and belly spinal neuron, the rat sciatic nerve CNTF that also have to purify it seems necrocytosis (the Sendtner et al. of motor neuron in the facial nerve (cranial nerve,seventh) that has prevented the neonate rat damage, 1990, Nature 345: 440-441).

Use recombinant DNA technique, the cloning and expression of CNTF has caused the active discovery of a series of CNTF.

2.3. growth factor receptors

A series of conducts cause that the acceptor of combination and response protein prime factor media had been characterized and molecular cloning in recent years, comprise the acceptor of Regular Insulin, platelet-derived somatomedin, Urogastron and correlative thereof, fibroblast growth factor and various interleukin and hemopoieticgrowth factor.Nearest data shown, some acceptor can with the combination of multiple (relevant) somatomedin, on and in other occasion, that the identical factor can act on is multiple (relevant) acceptor (Lupu et al. for example, 1990, Science 249: 1552-1555; Dionne et al., 1990, EMBO J.9: 2685-2692; Miki et al., 1991, Science 251: 72-75).The acceptor of most of conjugated protein prime factors can be characterized as being widely has born of the same parents' outside part that can respond binding factor specifically, cross over the transmembrane zone of cytolemma, with the intracellular region territory, this zone is often included in when rho factor is attached on born of the same parents' outside part of acceptor in the start signal transduction.Meaningfully, although many acceptors are made up of the single polypeptide chain, be attached on its factor but other acceptors obviously need (at least) two independent subunits with high affinity and can function after combination response (Hempstead et al. for example, 1989, Science 243: 373-375; Hibi et al., 1990, Cell 63: 1149-1157).The born of the same parents of given acceptor are outer and the inner branch of born of the same parents is frequent and other acceptor respective regions all has common Structural Theme (motif), represented not evolution between the isoacceptor and function relation.These relations often are quite far away and may fully reflect the repeated use of certain normal areas structure.For example, the multiple combination independently not isoacceptor of the factor utilizes " immunoglobulin (Ig) " zone in its born of the same parents' outside part, and other acceptors utilize " cytokine acceptor " zone in its factor calmodulin binding domain CaM (Akira et al. for example, 1990, The FASEB J.4: 2860-2867).The acceptor that has outer calmodulin binding domain CaM not born of the same parents (thereby this zone combines the different factors) in a large number (for example contains the promising relevant kytoplasm inner compartment that institute's activatory tyrosine characteristic protein kinase is encoded in the response factor combination, Ullrich and Schlessinger, 1990, Cell 61: 203-212).Factor combination is beyonded one's depth the mechanism of signal transduction process " activation ", even also is like this under the occasion of receptor tyrosine kinase.In other acceptors the intracellular region territory encode unknown function the zone or in conjunction with second kind of protein of component and unknown function link (Hibi et al. for example, 1990, Cell 63: 1149-1157), the activation of signal transduction even more indigestion like this.

3, summary of the invention

The present invention relates to CNTF acceptor (CNTFR) gene and protein.The clone and the characterization of human body CNTFR gene and expression thereof in the COS cell that it is based in part in transfection.

The invention provides nucleotide sequence and the deutero-fragment thus of coding CNTFR.The present invention also provides the CNTFR protein of purifying basically, with and peptide fragment.

Another aspect of the present invention is, can utilize the CNTFR probe, comprises nucleic acid and antibody probe, differentiates the molecule that CNTFR is relevant.For example, the invention provides such molecule, thereby they and CNTFR form complex body and participate in the CNTFR function.Another example is, the invention provides acceptor molecule, and they and CNTFR are homologous or cross reactivity on antigen, but inequality.These specific embodiments are based on such discovery, associated molecule has homology on CNTFR and other physiology, comprise: should should be mentioned that the IL-6 acceptor especially, but also have pdgf receptor, CSF-1 acceptor, prolactin antagonist acceptor, IL-2 and IL-4 acceptor, GM-CSF granular leukocyte macrophage colony stimulating factor receptor, pregnant special α 1-β glycoprotein and carcinomebryonic antigen-kind of tumor-marker.

The present invention also provides detection CNTF active analytical system, comprise expressing high-level CNTFR, thereby and in addition the CNTF or all highstrung cell of class CNTF molecule of extremely low concentration.

In addition, the invention provides to studying the test model system of CNTF physiological role.These systems comprise animal model, be exposed to animal in the circulation CNTFR peptide such as (ⅰ), thereby these peptides and cell receptor contention CNTF are in conjunction with the state that also causes the CNTF disappearance, (ⅱ) use the animal of CNTFR immunity, thereby (ⅲ) express high-level CNTFR also to CNTF transgenic animal hypersensitive; And (ⅳ) use stem cells technology deutero-animal, give birth to the CNTFR gene in wherein and be embryo by genomic deletion.

In another embodiment of the present invention, can use the CNTFR probe to differentiate cell and the tissue that responds normal or disease state CNTF.For example, the patient who suffers from the CNTF associated disorders can present the abnormality that CNTFR expresses.

In addition, CNTFR group of the present invention and protein can use in treatment.For example, but conduct does not limit, and can use circulation CNTFR to reduce the level of CNTF in the nervous system injury zone, center.On the other hand, recombinant chou CNTFR gene can be inserted in the tissue that has benefited from improving to CNTF sensitivity, as suffers the motor neuron among the amyotrophic side sclerosis patient.

4. description of drawings

The synoptic diagram of Fig. 1 .A. applying marking ligand combining method cloning by expression.B. secondary iodate antibody test has shown, with the comparison with the COS cell of primary cDNA storehouse transfection, many COS cells are taken turns the DNA transfection that obtains after the pan analysis (panning) with one and (carried out radioautogram on the 60mm of rotaring redyeing COS cell plate; Each blackspot represents to express the single rotaring redyeing COS cell of CNTF binding site).C. as (B) described identical check, but the COS cell with non-CNTFR coding plasmid (negative clone) or CNTFR plasmid (the just cloning) transfection of encoding.What illustrate only is the small portion of each plate.D. use the negative clone of (C) or just cloning the result with fluorescence amplifying cell separator (FACS) analysis of the COS cell of transfection.

Nucleotide sequence of Fig. 2 .CNTFR code cDNA (SEQ ID NO:1) and the aminoacid sequence (SEQ ID NO:2) of deriving.

Fig. 3. the arrangement of the human body CNTFR of the homology in demonstration immunoglobulin like protein zone and class cytokine acceptor zone.Left-hand digit is represented the amino acid number that begun by first methionine(Met).Residue that is equal to and the surrogate of reservation square frame mark.Insert the gap to reach maximum homology.IL-6=interleukin 6; The CEA=carcinomebryonic antigen, the somatomedin that PDGF=is platelet-derived, CSF-1=colony-stimulating factor 1; α 1-β GD=α 1 β glycoprotein, PRL=prolactin antagonist, EPO=erythropoietin; IL-2=interleukin 2; IL-4=interleukin 4, the GM-CSF=rHuGM-CSF.

Structural relation between Fig. 4 .CNTFR and other associated receptors.Human body IL-6 acceptor and CNTFR have the immunoglobulin (Ig) zone of a fusion on the factor calmodulin binding domain CaM end that is proposed.The factor calmodulin binding domain CaM that a sour chain of weak point (zigzag line) connects spherical immunoglobulin (Ig) and proposed.As discussed in this article, the protein that is similar to gp130 that is proposed demonstrates with CNTFR and connects.HuGRHR-human hormone acceptor; RbPRLR-exempts from the prolactin antagonist acceptor; MoEPOR-mouse erythropoietin receptor; Plain-2 acceptor beta chain between HuIL2R β-human leukocytes; Plain 6 acceptors between the HuIL6R-human leukocytes; HuCNTFR-human body ciliary deutero-human BDNF acceptor; The C-halfcystine; The X-unknown amino acid; The W-tryptophane; The S-Serine; The F-phenylalanine.

The tissue positioned of Fig. 5 .CNTFR message.As making RNA in the specified tissue by rat as described in the 8.1. joint.As constitute the dna fragmentation of deutero-CNTFR in the thing as described in the 8.1. joint by the expression that in pCMX, contains these groups.Tissue: cerebellum (CD); Hindbrain (HB); Midbrain (MB); Thalamus (TH/HYP); Striatum (STRI); Hippocampus B(HIP B); Hippocampus A(HIP A); Cortex (CORT); Olfactory bulb (OLF); Adult brain (ADBR); Skin (SK); Heart (HRT); Muscle (MUS); Lung (LUNG); Intestines (INT); Kidney (KID); Liver (LIV); Spleen (SPL); Thymus gland (THY); E17 liver (E17LIV).

Fig. 6. contain the pCMX of hCNTF-R group inset.The formation of pCMX in the application of awaiting the reply jointly.

Fig. 7. the Northern engram analysis of CNTF expression of receptor in the skeletal muscle.The total RNA of test 10 μ g in each row.Row 1, mouse muscle-forming cell are C2C12mb; Row 2, mouse tubulose myocyte is C2C12mt; Row 3, rat tubulose myocyte is C2C12mt; Row 4, rat tubulose myocyte is L6mt; Row 5, rat soleus muscle; Row 6, rat toe length is stretched (EDL) flesh; Row 7, Denervated skeletal muscle; Row 8, the human body tubulose flesh of purification; Row 9, skeletal muscle (this RNA sample is degraded); Row 10, the adult rat cerebellum; Row 11, the sham-operation soleus muscle; 12,72 hours Denervated rat soleus muscles of row; Row 13, the EDL flesh of sham-operation; 14,72 hours Denervated EDL flesh of row.

Fig. 8. stand the internal anatomy of the right hind of surgery neurectomy.

Fig. 9 .UNOP=is from animal groups 1(table IV) soleus muscle of operation not; On the left of solid bars represents that right side and strokes and dots bar are represented; NONE=does not pass through any injection liquid from (Denervated, the right side) of the damage of animal groups 2 and contrast (sham-operation) soleus muscle; ALB=uses PBS/BSA(SC from animal groups 6) (right side) of the damage handled and contrast (left side) soleus muscle; CNTF=uses CNTF/BSA(SC from animal groups 5) (right side) of the damage handled and contrast (left side) soleus muscle.Solid bars: damage; Strokes and dots bar: contrast.

5. Fa Ming detailed description

In order clearly to describe the present invention, but be not as limiting, detailed description of the present invention will be divided into following a few part:

(ⅰ) clone CNTF acceptor;

(ⅱ) nucleic acid encoding CNTF acceptor;

(ⅲ) CNTFR peptide;

(ⅳ) CNTF receptor expression;

(ⅴ) differentiate the molecule relevant with the CNTF acceptor; And

(ⅵ) application of the present invention.

5.1. clone's the ciliary neurotrophic factor receptor

The present invention can be by providing a kind of method clone CNTF acceptor (CNTFR) of selecting to express the target cell of CNTFR.By a kind of means of enrichment CNTFR encoding sequence are provided, the present invention can purify CNTFR protein and direct clone CNTFR coding DNA.

For example, can select to contain the target cell of CNTFR, and CNTFR protein can use the method for the known purification acceptor molecule of this professional domain those of ordinary skill to purify.For example, but be not as limiting, as CNTF as described in following 5,6.3 joints or attached to the CNTF(CNTF/ mark on the detectable molecule), can be reversibly crosslinked on target cell, wherein mark can be radio-labeling, antigenic determinant or antibody, only lifts several examples here; And can purify from the proteinic cytolemma of described target cell connecting.These methods of purification can comprise SDS-PAGE, and then detect the position of CNTF in the gel or CNTF/ mark; For example, can use radiolabeled CNTF, and be linked on its acceptor, can in gel, be observed by radioautogram.On the other hand, can in the Western engram technology, use anti-CNTF or anti-tag antibody to identify the position of the CNTF/ acceptor complex body in these gels.Can utilize protein that preliminary gel electrophoresis separates q.s with the amino acid sequencing of the peptide fragment of realizing acceptor or allow to prepare can be in order to the anti-CNTFR antibody of CNTFR molecule in the purification target cell extract.The aminoacid sequence that is obtained by the CNTFR that purifies can be used for designing degeneracy oligonucleotide probe, and these probes can be used to identify CNTFR coding cloned genomic dna storehouse or preferably be cloned into the nucleic acid in the cDNA storehouse that a target cell that is produced by CNTFR constitutes.

On the other hand, CNTFR can be cloned by the subtractive hybridization method, wherein mRNA can be by the target cell preparation of expressing CNTFR, and then can by make mRNA(or by its cDNA that makes) with by the cell that can not express CNTFR, as the non-CNTFR encoding sequence of neuronal cell deutero-mRNA or cDNA hybridization deduction.The remaining nucleic acid in deduction back can easily be enriched in the CNTFR encoding sequence.

Preferably, by the endogenous expression of CNTFR and/or by the deduction technology of discussing as mentioned above, the nucleic acid that the target cell from be enriched in the CNTFR encoding sequence makes also can be used in the cloning by expression technology with direct clone CNTFR.For example, but not as limiting, can prepare from the total genomic dna of the target cell of expressing CNTFR and subsequently it is transfected into can not express CNTFR and preferably by in the different plant species institute deutero-clone in the target cell species (for example, DNA from human body CNTFR Codocyte can be transfected into mouse cell, in a kind of L cell).Although the transfectional cell of lesser amt can be expressed CNTFR, these cells can by as hereinafter 5,6.3. joint described growing thickly (rosetting) technology or immunofluorescence technique is identified and can by the known fluorescence amplifying cell separator of those of ordinary skill in this professional domain for example utilize the antibody coupling magnetic bead or pan analysis (panning) technical point from.Can be by preparing genomic library by transfectant, separate then and breed these and both contained sequence with the CNTFR consensus amino acid sequence, contain the clone with species specific gene element homologous sequence again, thus clone CNTFR coding DNA in the transfectant that produces by acceptor; For example, can identify human DNA by the Alu repeated sequence that in the whole human body genome, distributes with high frequency.For example, but not as limiting, can select to breed the non-human body cell of cultivation of the human DNA of the transfection that contains coding CNTFR and express human body CNTFR, the non-human body cell that can utilize the genomic dna transfection that makes by these cells to cultivate then, and can select the CNTFR express cell.This technology can repeat; Its objective is with each transfection step to reduce the amount of the human DNA that in the CNTFR Codocyte, exists.Therefore, when the genomic dna of the human body CNTFR of the express cell of transfection is cloned when producing information storage, the clone who comprises human DNA (being identified, for example by tangible human body sequential element is screened) when carrying out the repetition transfection more may contain the CNTFR encoding sequence.

Can be introduced in XenoPus (Xenopus) ovocyte with the aggregation form by direct injection from the RNA of CNTFR express cell element or tissue source or by the cDNA expression library that this source obtains; Can be with the ovocyte that the aggregation of coding CNTFR is injected by being identified to the detection (for example ionic current) of function response, the function response can be by being exposed to these ovocytes among the CNTF, and perhaps another kind of method is induced by detect the CNTF binding site that exists on the ovocyte surface of these injections.Repeatedly the aggregation of determining is divided into the individuality clone that more and more littler aggregation can cause identification code CNTFR.

On the other hand, the cDNA expression library can be derived and is used to subsequently during transient expression detects by the CNTFR that is loaded with target cell.In a preferable embodiment of the present invention, described expression library can and use the COS cell to carry out transient expression and detect in conjunction with the SV40 copy source.Can examine and determine CNTFR as mentioned above and express transfectant, and can use standard method to recover the CNTFR coding DNA.Can use then as the described method of nucleic acid encoding CNTF being bred the nucleotide sequence of coding CNTFR and/or using it in the expression system, is 07/,570 651 as people such as Sendtner at application number, name is called " Ciliary Neurotrophic Factor ", and the applying date is described in the U.S. Patent application in August 20 nineteen ninety.

6. save in the illustrational particular of the present invention of (see figure 1)s hereinafter, can be as the following cloning by expression that carries out CNTFR.Can be by the clone or the tissue preparation cDNA storehouse of expressing CNTFR such as SH-SY5Y, so that cDNA is inserted in the expression vector.For example can use then that DEAE/ chloroquine transfection operative technique is transfected into this storehouse in the suitable clone, in COS M5 cell.After the transfection several days, these cells can be separated and make it to carry out the Aruffo/Seed pan from its culture dish and analyze (panning) step (Seed and Aruffo, 1987, Proc.Natl.Acad.Sci.U.S.A.84: 365-3369), following change is arranged simultaneously:

(ⅰ) can be at first with the CNTF(of mark CNTF fungi (myc) for example) these cells are being cultivated on ice about 30 minutes, by phosphate buffered saline (PBS) (PBS)/2%Ficoll centrifugation to remove excessive ligand, use anti-tag antibody (for example anti fungal antibody 9E10) to cultivate on ice then about 30 minutes, rather than cultivate cells transfected with antireceptor antibody.

(ⅱ) then can be with these cells by the PBS/2%Ficoll centrifugation and subsequently on the flat board that scribbles the antibody (, then being anti-mouse antibodies for example) that to discern anti-tag antibody " pan analysis (panned) " if anti-tag antibody is 9E10.

Then,, can make the Hirt supernatant liquor by adherent cell, and can have sinking shallow lake plasmid DNA not behind the adherent cell at flush away from flat board at the 10-20 μ g tRNA that has an appointment.Can pass through standard technique then, include but not limited to, electroporation is introduced the plasmid DNA that obtains in the suitable bacterium (for example DH10 B bacterium).Culture by the bacterial growth that transforms can be used for preparing the plasmid DNA that another takes turns eukaryotic cell transfection and pan washing then.After the transfection second time, pan washing and plasmid DNA preparation and transforming, can on selective medium, turn out the bacterium transformant by plate, can collect individual bacterium colony and be used to prepare plasmid DNA, the DNA that is made by a series of these clones can be respectively applied for the COS cell transfecting.On the other hand, before the individual bacterium colony of test, may need the inferior enrichment of more wheels.Can include but not limited to that the direct bond test of use radioactivation mark or fluorescent mark indicator antibody can be identified the COS cell of the expression CNTF binding site of generation by a series of technology.The example of a CNTFR coding nucleic acid is included in the pCMX-hCNTFR(I 2 of Fig. 6) in, it is deposited in NRRL, preserving number is B-18789, and be disclosed in application number by Davis and Yan-copoulos and be, name is called in the U.S. Patent application of " Mammalin Expres-sion Vector ".Then can be by the clone that restriction fragment is drawn and the analysis of nucleic acid sequencing is differentiated with this method who uses standard technique.Can utilize the standard hybridization technique to use CNTFR code cDNA fragment for example to contain genomic dna sequence then from the CNTFR gene in genomic dna storehouse with evaluation.

In case after obtaining, can utilize any known method clone or subclone CNTFR gene in the prior art.Can use a large amount of carrier-host system well known in the prior art.Possible carrier includes, but not limited to the virus of clay, plasmid or variation, but this carrier system must can be affine with the host cell that uses.These carriers include, but not limited to phage such as the λ derivative, or plasmid such as pBR322, pUC or Blue script

(Stratagene) plasmid derivative thing can be introduced host cell with the recombinant chou molecule by conversion, transfection, infection, electroporation etc.

The CNTFR gene can be inserted can be used to transform, transfection or infect in the cloning vector of proper host cell, so that produce many copy gene orders.This can realize by dna fragmentation is connected in the cloning vector with additional sticky end.Yet, not being used for DNA is divided into segmental additional restriction site if in cloning vector, do not exist, the end of dna molecular can be modified on enzymic activity.It has proved that restriction endonuclease is divided the site to be attached on the oligonucleotide primer that is used for the polymerase chain reaction to promote to be inserted into this advantage of carrier.On the other hand, can make required any site on the DNA end by nucleotide sequence (linker) is connected to; These connect the restriction endonuclease recognition sequence that linker can comprise the oligonucleotide coding of specific chemosynthesis.In a selectable method, can modify splitted carrier and CNTFR gene by the homopolymerization tailing.

In specific embodiment, the conversion that contains in conjunction with the host cell of the recombinant DNA molecules of a kind of isolating CNTFR gene, cDNA or synthetic dna sequence dna can produce a plurality of copy genes.Thereby, by the growth transformant, from transformant, separate recombinant DNA molecules, and from isolating recombinant DNA, regain the gene that inserts in case of necessity, can obtain this gene in a large number.

5.2. nucleic acid encoding the ciliary neurotrophic factor receptor

Method among the embodiment that utilization is above described in detail and hereinafter 6. joints has been measured following nucleotide sequence, and the amino acid sequence corresponding of having derived.Described the sequence (SEQ ID NO:1) of human body CNTFR among Fig. 2.Can use this sequence according to the present invention, its functional equivalence thing, or length is at least the fragment of this sequence of 6 Nucleotide.In addition, the present invention relates to by isolated CNTFR gene in pig, sheep, feline, birds, horse or dog and primates source and the active species of any CNTF of existence.But the subsequence that contains the hybridization portion of CNTFR sequence can be used, for example, in nucleic acid hybridization detection, Southern and the Northern engram analysis etc.

For example, the nucleotide sequence of describing among Fig. 2 (SEQ ID NO:1) can change as replacing, add or lacking by the sudden change of the first-class valency molecule sequence of coding function is provided.According to the present invention, if a kind of molecule has the ability in conjunction with CNTF, then this molecule is of equal value or active on the function with the molecular ratio with Fig. 2 (SEQ ID NO:1) sequence of describing, but it needn't be in conjunction with the CNTF with avidity similar to natural CNTFR.Because the degeneration of nucleotide coding sequence, in practice of the present invention, can use coding basically as other dna sequence dnas of the same acid sequence described among Fig. 2 (SEQ ID NO:2).These sequences comprise, but be not limited to contain the nucleotide sequence of the CNTFR gene of being described among all or part of Fig. 2 (SEQ ID NO:2), this gene is reformed by the different codons that are substituted in the amino-acid residue of the first-class valency of coding function in the sequence scope, thereby produces the outage variation.

In addition, can design recombinant chou CNTFR nucleic acid sequence encoding of the present invention dexterously, so that modify processing and the expression of CNTFR.For example, but not that the CNTFR gene can combine with promoter sequence and/or ribosome binding site as qualification, perhaps can insert signal sequence so that secretion CNTFR also promotes to gather in the crops or bioavailability thus in CNTFR encoding sequence upstream.

In addition, given CNTFR can translate, start and/or terminator sequence with foundation and/or destruction at external or vivo mutations, perhaps in coding range, set up varient and/or form new restriction endonuclease site or site that destruction is pre-existing in, to promote external further modification.Can use any induced-mutation technique well known in the prior art, include but not limited to, external locating point mutagenesis (Hutchinson et al., 1978, J.Biol.Chem.253: 6551), use TAB

Linker (Pharmacia) etc.

5.3. the ciliary neurotrophic factor receptor peptide

The present invention also provides CNTFR protein, fragment and derivative thereof or its functional equivalence thing of the aminoacid sequence that has above-mentioned Fig. 2 in (SEQ ID NO:2) and protein (this homology is at least about 30%) with this protein homology is provided.The present invention provides also that to comprise at least 6 amino acid, comprise on a kind of antigenic determinant or the function be proteinic fragment of active CNTFR or derivative.CNTFR protein molecular weight with aminoacid sequence that Fig. 2 (SEQ ID NO:2) described is about 42kd.

CNTFR protein of the present invention or fragment or derivatives thereof comprise, but be not limited to, contain all or part ofly as original amino acid, comprise wherein with amino-acid residue of equal value on the function replacing the change sequence that the residue in the sequence causes outage to change basically as the material of the aminoacid sequence that Fig. 2 described.For example, the another kind of similar polar amino acid that one or more amino-acid residues in the sequence scope are used as the functional equivalence thing replaces, and causes the outage change.Can be by the amino acid whose substituent of selecting among the member of class under other amino acid in the sequence scope.For example, the amino acid of nonpolar (hydrophobic) comprises L-Ala, leucine, Isoleucine, Xie Ansuan, proline(Pro), phenylalanine, tryptophane and methionine(Met).Polar neutral amino acid comprises glycine, Serine, Threonine, halfcystine, tyrosine, asparagus fern amino acid and glu famine.Positive charge (alkalescence) amino acid comprises arginine, Methionin and Histidine.Negative charge (acidity) amino acid comprises aspartic acid and L-glutamic acid.For example also comprise by phosphorylation, glycosylation, crosslinked, acidylate, proteolysis division within the scope of the invention, be bonded on a kind of antibody molecule, membrane molecule or other ligands; CNTFR protein or fragment or derivatives thereof (the Ferguson et al. that modifies respectively during translating or afterwards; 1988, Ann.Rev.Biochem.57: 285-320).

CNTFR peptide of the present invention can make by the recombinant nucleic acid expression technology or by the synthetic technology chemosynthesis that utilizes standard peptide.

5.4. the expression of the ciliary neurotrophic factor receptor

For expression recombinant CNTFR, to the nucleotide sequence of CNTFR protein coding, or its part can be inserted in the suitable expression vector promptly a kind of carrier that contains the bioelement of transcribing and translating this insertion protein coding sequence.Necessary transcribe and translate signal and also can provide by natural CNTFR gene and/or its side regions.Can use multiple host-vector system with the marking protein encoding sequence.These systems include, but not limited to the mammal cell line system of infective virus (for example, vaccinia virus, adenovirus); The insect cell system of infective virus (for example baculovirus); Microorganism, as the yeast that contains yeast vector or the bacterium that transforms with phage DNA, plasmid DNA or cosmid DNA.The expressive element of these carriers is difference aspect its intensity and specificity.According to used host-vector system, can use to appoint-series suitable transcribe and translate element.

In a preferable specific embodiments of the present invention, the CNTFR gene can be included in be deposited among the NRRL, in the pCMX expression vector that specified preserving number is No.B-18790.

Can use to dna fragmentation being inserted above-mentioned any method in the carrier contains the mosaic gene of being made up of transcription/translation control signal and protein coding sequence with formation expression vector.These methods can comprise reorganization (gene recombination) in vitro recombination body DNA and synthetic technology and the body.The expression of the nucleotide sequence of coding CNTFR protein or peptide fragment can be regulated so that CNTFR protein or peptide are expressed in the recombinant DNA molecules host transformed by second kind of nucleotide sequence.For example, can be by the expression of any promotor well known in the prior art/intensifying factor control CNTFR.Can be used to control the CNTFR expression promoter comprises, but be not limited to, SV40 early promoter zone (Bernoist and Cham-bon, 1981, Nature 290: 304-310), the CMV promotor, contained promotor (Yamamoto in 3 ' end of Rous sarcoma virus duplicates, et al., 1980, Cell 22:787-797), herpes thymidine kinase promoter (Wagner et al., 1981, Proc.Natl.Acad.Sci.U.S.A.78: 144-1445), the adjusting sequence of metal thionin gene (Brinster et al., 1982, Nature 296: 39-42); Prokaryotic expression carrier, as β-Nei Xiananmei promotor (Villa-Kamaroff, et al., 1978, Proc.Natl.Acad.Sci.U.S.A.75: 3727-3731), or tac promotor (DeBoer, et al., 1983, Proc.Natl.Acad.Sci.U.S.A.80: 21-25), also can be referring to " Useful proteins from recombinant bacteria " in Scientific American, 1980,242: 74-94; Promotor from yeast or other fungies, as Ga14 promotor, ADC(alcoholdehydrogenase) promotor, PGK(phosphoglyceric kinase) promotor, alkaline phosphatase promoter and below present organizing specific and be used for the animal transcripting controling areas of transgenic animal: at the active elastoser I of pancreas cystencyte gene-controlled area (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986 Cold Spring Harbor Symp.Quant.Biol.50: 399-409; MacDonald, 1987, Hepa-tology 7: 425-515); Active insulin gene control region in pancreas β cell (Hana-han, 1985, Nature 315: 115-122), active immunoglobulin gene control region in lymphoidocyte (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol.Cell.Biol.7: 1436-1444), active mouse virolactia control region (Leder et al. in testis, chest, lymph sample and mastocyte, 1986, Cell 45: 485-495), active albumin gene control region (Pinkert et al. in liver, 1987, Genes and Devel.1: 268-276), active α-fetoprotein gene-controlled area (Krumlanf et al., 1985, Mol.Cell.Biol.5: 1639-1648 in liver; Hammer et al., 1987, Science 235: 53-58); Active alpha1-antitrypsin gene-controlled area (Kelsey et al. in liver, 1987, Genes and Devel.1: 161-171), active beta-globin gene-controlled area (Mogram et al. in the myelin body cell, 1985, Nature 315: 338-340; Kollias et al., 1986 Cell 46: 89-94), active myelin basic protein gene-controlled area in the oligodendroglia of brain (Readhead et al., 1987, Cell 48: 703-712); Active myosin light chain-2 gene-controlled area (Sani in skeletal muscle, 1985, Nature 314: 283-286), and in hypothalamus active gonadotropin releasing hormone gene-controlled area (Mason et al., 1986, Science 234: 1372-1378).

The expression vector that contains CNTFR gene insertion body can be identified with three usual ways:

(a) DNA-DNA hybridization, (b) functional existence of " mark " gene or disappearance, and (c) expression of insertion sequence.At first, the existence that is inserted into the heterologous gene in the expression vector can contain probe with the CNTFR base homologous sequence that inserts by use and carries out DNA-DNA and hybridize and survey.In second step, recombinant vectors/host system can be according to owing to be inserted into heterologous gene existence that some " mark " gene official of causing in the carrier can (for example thymidine kinase activity antibiotics resistance, the phenotype that makes the transition, the formation etc. of closed shape (occlusion body) in baculovirus) or disappearance and identify and select.For example, if the CNTFR gene is inserted into the marker gene sequence inside of carrier, contains the functional disappearance of recombinant chou serviceable indicia gene that CNTFR inserts body and identify.In the 3rd step, recombinant expression vector can be identified by analyzing the heterologous gene products of being expressed by recombinant chou.This analysis can for example upward or on the antibody of Direct Recognition CNTFR be dependent on for example physics or the functionality of CNTFR gene product by making acceptor be connected to CNTF.

In another embodiment, cell that can not normal expression CNTFR can with the nucleic acid transfection of recombinant C NTFR coding then by transfectant is exposed to CNTFR down and the increase of measuring the cAMP level subsequently measure the expression that the official can CNTFR.

In case specific recombinant DNA molecules is identified and is separated, and just can use several method known in the art to make it breeding.In case set up a suitable host system and growth conditions, recombinant expression vector just can breed and prepare in large quantities.As previously mentioned, operable expression vector comprises following carrier or derivatives thereof: human or animal's virus, such as vaccinia virus or adenovirus; Insect viruses are such as baculovirus; Yeast vector; Phage vector (for example λ), and plasmid and cosmid DNA carrier are only lifted several examples here, are not limited to these.

In addition, can select a kind of host cell strain so that adjust the expression of insertion sequence or modify also processed gene product in the concrete mode of hope.Can in the presence of some inductor, be improved from the expression of some specific promotor; Thereby the CNTFR protein expression of geneization can Be Controlled.And different host cells have its specific and concrete mechanism to carry out proteinic processing and modification (for example glycosylation, division) after translating and translating.τ is to select suitable clone or host system to guarantee that the foreign protein matter of being expressed is carried out desirable modification and processing.For example, can use expression in the bacterium system to produce not glycosylated core protein product.Expression in yeast can be used for producing glycosylated product.Expression in mammalian cell can be used for guaranteeing xenogenesis CNTFR proteinic " natural " glycosylation.And different carriers/host expresses system can make the processing reaction proceed to different degree such as the proteolysis division.

In case a kind of recombinant chou of the CNTFR of expression gene is identified, just should carry out the analysis of gene product.This can be by realizing based on the physics of product or functionality's analysis.

In case CNTFR protein is identified, just can utilize following standard method with it separation and purifying, these standard methods comprise the standard method of chromatography (for example ion-exchange, affinity and fractionation column chromatography), centrifuging, differential solubility method or any other protein purification.Particularly, CNTFR protein can contain the affinity column that is connected to a CNTF on the fixed support and separates by being connected to one.

CNTFR or its functional activity part complementary nucleotide sequence have also been prepared with DNA or RNA sequence encoding.A kind of particular case is, can synthesize anti-meaning oligonucleotide, and it and at least a portion CNTFR mRNA are complementary.

5.5. the evaluation of the molecule relevant with cntf receptor

Defined a plurality of acceptor factor systems, the wherein same factor can be connected to (as indicated above) on a plurality of acceptors.To the CNTF situation also is so, and the present invention can be by identifying any other CNTFR acceptor with being used to obtain the identical step of CNTFR described herein, but is used to prepare except the RNA source of cDNA expression library.Can select easily to express the source of different CNTF acceptors; Can in the presence of connecting, the CNTF that CNTFR is not had effect assess (gene probe that produces from the CNTFR sequence can be used for the protein and the CNTF described here that cause CNTF to be connected are compared with antibody reagent) clone or tissue source to the source.In addition, because known receptor was connected to more than (as indicated above) on one the correlation factor, the evaluation of CNTFR should be able to identify that also any other is connected to natural ligand on this receptor.

Another aspect of the present invention is that the CNTFR sequence can be used for identifying the molecule relevant with CNTFR.CNTFR contains and the common theme of multiple other acceptor.The extracellular part of CNTFR had not only contained " immunoglobulin (Ig) " territory but also had contained " cytokine acceptor " zone at its N-terminal, and the two is by short stranded distinguishing.Although many receptor homologs, only have a kind of acceptor-IL-6 acceptor-have the particular arrangement in these identical territories with CNTFR in " immunoglobulin (Ig) " or " cytokine acceptor ".Therefore the IL-6 acceptor is and the maximally related protein of CNTFR.Interesting is, the IL-6 acceptor has a very short kytoplasm internal area, and this and CNTFR also are similarly, this kytoplasm internal area significantly to start the IL-6 connection response be unnecessary (Hibi et al., 1990, Cell 63: 1149-1157).Recently, a kind of novel signal transductant of IL-6 acceptor is called gp130, by molecular cloning.Itself does not connect IL-6 this transductant, but it really gives the high-affinity connection for the IL-6 acceptor, and need it transduce the IL-6 signal (Hibi et al., 1990, Cell 63: 1149-1157).The clone of CNTFR shows that it and IL-6 acceptor have the common key character, and these features are not discovery in other known receptor, thereby has defined a new family of acceptor.Defined as the present invention, preceding two members' homology can be used to identify other associated receptor through following method in this receptor family, promptly utilize DNA or corresponding to the antibody probe of homologous region, perhaps use polymerase chain reaction method simultaneously and corresponding to the degeneracy oligonucleotide of the consensus of amino acid identity (Maisonpierre et al. for example, 1990, Science 247: 1146-1451).The present invention also can be used for testing CNTFR and whether uses the signal transduction body identical with the IL-6 acceptor, and perhaps whether it uses relevant molecule.At last, the evaluation of the acceptor that CNTFR is relevant helps the evaluation of the novel ligand that is connected with these acceptors.

According to the present invention, by using oligonucleotide screening DNA storehouse corresponding to CNTFR sequence shown in Fig. 2 (SEQ ID No:1) or that derive from protein sequence data or from nucleotide sequence (comprise genomic dna or be cDNA better), so that can be identified for the clone of newcomer's coding of above-mentioned family.By reducing the severity of hybridization, the evaluator to this differentiated member of family part omitted can be increased.Also wish to use basically the common sequence of member in this family with sequencing; These height conservation zones are useful especially during other member in identifying this family.The screening in storehouse can be by using for example Benton and Davis(1977, Science 196: 180) or Grunstein and Hogness(1975, Proc.Natl.Acad.Sci.U.S.A.72: hybridization technique 3961-3965) is carried out.Can further analyze the clone who identifies with hybridization then, and the newcomer can identify by restriction fragment drawing (mapping) and sequencing technology according to method well known in the art.

Hope use polymerase chain reaction (PCR) technology (Saiki et al., 1985, Science 230: 1350-1354) identify other member in the total family of CNTFR.For example, can be used among the PCR, be preferably and use cDNA as template corresponding to having a mind to of known CNTFR sequence and anti-meaning primer.Wish these primers of preparation so that make it to comprise Restriction Enzyme division site, these division sites can promote the product of PCR is inserted in the appropriate carriers can select resulting clone to the newcomer then.This selection can use standard technique to realize, comprises the hybridization analysis of using corresponding to the probe of known array.For example, as mentioned above, a series of probes of the proteinic not same district of CNTFR of a kind of characterization of expression are being hybridised on the clone's who is loaded with free PCR generation the bilayer filter under the low severity.Observe, various clones can hybridize some probe but can not hybridize other probes.The newcomer also can be identified that wherein different with the probe that derives newcomers can be hybridized not too strongly by the severity that increases hybridization conditions in the family under high severity from known member.In addition, the newcomer can use standard technique to identify with respect to known member's difference to disclose restricted figure or sequence by restricted drawing or sequencing analysis in the family.

In another embodiment, thus the invention provides and form title complex with CNTFR and participate in the functional molecule of CNTFR.For example, have found that by sequential analysis, CNTFR does not demonstrate has the kytoplasm zone; In fact, it can be attached to (at Ferguson et al., 1988, Ann.Rev.Biochem.57: be described among the 285-320) on the film by GPI key glycosyl-phosphatidyl inositol.This shows that at least a other molecule and CNTFR have formed association and participates in signal transduction by cytolemma.A kind of like this molecule can be a kind of protein of for example following IL-6R to find together, such as GP130(Taga et al., and 1989, Cell 58: 573-581); From this situation of homology between CNTFR and the IL-6 acceptor is possible especially.The molecule that forms association with CNTFR on cytolemma can separate and identifies with any methods known in the art, comprise chemically crosslinked, with the co-precipitation of anti-CNTFR antibody or use the CNTF/ mark and/or use protein or lipoid purification techniques, but be not limited to these methods.

In addition, the invention provides the molecule that can be connected except that CNTF with CNTFR.These molecules are defined as the molecule that is connected with CNTF competition CNTFR, comprise other conventional ligand, and these molecules comprise peptide, peptide derivant and non-peptide (for example class peptide) compound

5.6. purposes of the present invention

5.6.1. mensuration system

The invention provides the mensuration system, therein can the physiological responses to CNTF detects owing to be exposed to the CNTF activity that produces in peptide or the non-peptide compound or be similar to the active activity of CNTF in response to the cell of the expression of CNTF CNTF molecule of the present invention or clone by measuring.Physiological responses comprises any physiological effect of CNTF; comprise top 2.2 joints described, and the transcription activating (for example promotor/enhanser and structure gene) of some nucleotide sequence, the processing relevant with CNTF, translate or phosphorylization, in response to directly or indirectly by the secondary process of CNTF inductive process induce and morphologic variation, such as the aixs cylinder budding or keep the ability that cell is survived such as ciliary nerves ganglion cell, motor neuron, purkinje's cell or hippocampal neuron; here only lift several examples, be not limited thereto.

In the preferable specific embodiments of the present invention, the functional response between CNTF and the CNTFR can observe by the increase that detects the turnout of the first responsive genes of activatory " early stage at once " when responding the transmembrane signal (including but not limited to c-fos and c-jun) of many factors stimulated growth.For example, the activation of immediate early gene can detect by the Northern engram analysis of immediate early gene mRNA level.In the preferable embodiment of the present invention, c-fos or c-jun mRNA level can be by determining that by the Northern engram analysis of the mRNA of the target cell preparation of cultivating with CNTF wherein the CNTF activity confirms with the increase of c-fos or c-jun level.It should be noted that, in a certain particular of the present invention, in case produce the target cell that contains recombinant C NTFR coding nucleic acid or select, just wish to guarantee that target cell characteristic ground is in response to CNTF or have the active compound of class CNTF by being connected with CNTF.In description of the invention, but term class CNTF activity is meant the active similar biological activity that can be the same or different with CNTF; These activity include but not limited to describe in above-mentioned 2.2 joints that perhaps concrete early promoter at once is such as the activation of fos or jun promotor.

The invention provides novel being used to and screen the mensuration system of CNTF activity or the active compound of class CNTF.The target cell that is connected on the CNTF can identify and separate by the precipitation of producing with the nucleic acid transfection of CNTFR coding and can be by for example fluorescence amplifying cell separator, rosette cell or restricted dilution, as described in 5.6.3 joint hereinafter.

In case target cell system is produced and identifies, just wish to select unusual responsive cell to CNTF.These target cells can have a large amount of CNTFR; The target cell that has a large amount of CNTFR can be identified by the target cell of selecting to be connected on the high-level CNTF, for example just produces the sizable fluorescence of quantity when cell is cultivated with the CNTF/ mark and carried out immunofluorescence analysis.In addition, when connecting, response CNTF can demonstrate great biological response to the unusual responsive cell of CNTF, such as the unexpected increase of immediate early gene product such as c-fos or c-jun.By improve using the suitable mensuration system of responsive target cell to CNTF, the invention provides screening CNTF activity or the active method of class CNTF, this method can detect low-level CNTF activity.

Particularly, use recombinant DNA technology, the invention provides and be designed to extremely sensitive CNTF target cell CNTF.For example, the methods clone's who describes according to 5.1 joints CNTF-acceptor genes can be inserted in the cell with natural CNTF response, and recombinant C NTFR gene is expressed with high level and the target cell of resulting design has been expressed a large amount of CNTFR on its cell surface as a result.

In other words, perhaps as another kind of situation, target cell can be designed to contain when response CNTF/ acceptor connects by the recombination of high level expression.This recombination is preferably the product that is attended by easy detection.For example, but and be limited to this, be used in the expression of control reporter gene in the structure that can be incorporated in the target cell from the transcripting controling area (being promotor/enhancing subarea) of immediate early gene.Immediate early gene/reporter gene makes up when passing through strong promoter/enhanser or high copy number order and during with high level expression, just can be used for producing connecting in response to CNTFR of a kind of amplification in target cell.For example; but be not limited to this; promotor (such as c-fos or c-jun promotor) with CNTF response can be used for controlling the expression of detectable reporter gene, and reporter gene comprises that beta-galactosidase enzymes, tethelin, chloramphenicol acetyltransferase, neomycin phosphoric acid change enzyme acetyl, luciferase or beta-glucuronidase.The detection of these reporter gene products is known those of ordinary skill in the art, and this detection can be used as the CNTF activity or the active sensitive indicators of class CNTF of pharmaceutical compound.

Above-mentioned CNTFR coding or reporter gene make up and can be inserted in the target cell with any method known in the art.These methods include but not limited to: transfection, electroporation, calcium phosphate/deae dextran method and cell spray gun and have of the production of above-mentioned structure as genetically modified transgenic animal, and from wherein using above-mentioned method to select the CNTF target cell.

Mensuration of the present invention system makes can screen the pharmaceutical compound that is used for the treatment of with the CNTF relative disease effectively.For example, but not, wish the pharmaceutical reagent that screening CNTF is active and cerebellum degeneration is had result of treatment as qualification.In the specific embodiments of the present invention, the purkinje's cell of response CNTF can be identified and be separated, and cultivates in little # of the foster plate of multiwell tissue culture then.The substratum that has added test reagent or added CNTF can join in the well with suitable contrast by infinite dilution.The existence, aixs cylinder budding etc. that detect cell then are improved, but and confirmed test reagent and CNTF active and their relative reactivity.Another example is to have been found that the motor neuron focus is optimum response (Sendtner et al., 1990, Nature 345: 440) to CNTF.Therefore wish to identify the class CNTF compound that can prevent motor neuron death afterwards as CNTF in axial surgical blanking (axotomy).Motor neuron with CNTF response can be used on the compound that can be used for treating motor neurone disease in the mensuration system with evaluation.Have been found that CNTF can axially prevent the motor neuron necrocytosis after the surgical blanking effectively, when attempt treatment Spinal injury, myatrophy adnation sclerosis and diabetic neuropathy, when design can be treated the medicine of these imbalances effectively, comprise that above-mentioned discovery clearly is the discovery of particularly important by the needed medicine of blood brain barrier layer.In view of above-mentioned discovery, key is to find a kind of reliable and responsive screening system of as method provided by the invention, another example is, if find that a kind of disease specific is relevant with the cranky CNTF response in the concrete tissue, the rational therapy to disease is to provide external CNTF for the patient so.Yet it is longer than endogenous CNTF to wish to work out the transformation period, perhaps as the CNTF stimulant, perhaps with the molecule of a concrete tissue as target.Therefore, method of the present invention can be used for producing the screening system that can be used in the effective and sensitivity of identifying the molecule with desirable character.Similar mensuration system can be used for identifying the CNTF antagonist

5.6.2. experimental model system

The present invention also provides the experimental model system of the physiological action that is used to study CNTF.In these model systems, CNTFR protein, peptide fragment or derivatives thereof can or join in this system or in this system and prepare.These model systems can be used for studying the influence that CNTF is excessive or CNTF lacks.These experimental model systems can be used for studying in the cell or tissue substratum, in whole animal body, particularly in the cell or tissue of whole animal or in the tissue culturing system or CNTFR express can be brought out or the embodiment of improved adjusting promotor control in a concrete timed interval (comprising embryo's generating process) under to the influence of the response of CNTF enhanced or reduction.In the specific embodiments of the present invention, the CMV promotor can be used for being controlled at the expression of CNTFR in the transgenic animal.Term " transgenic animal " is meant the non-human transgenic animal herein, comprise the transgenosis mosaic, the non-human transgenic animal has transgenosis in its some or all cell, it comprises any inhuman species, and available any method production as known in the art, said method includes but not limited to: microinjection, cytogamy, transfection, electroporation, etc.For example, animal can produce with the zygote micro-injection method, such as what describe in " Brinster et al., 1989, Proc.Natl.Acad.Sci.U.S.A.82: 4438-4442 ".

The present invention also provides autoimmunization therein to correspond directly to the model system of CNTFR to autoimmune disease.Comprised with the CNTFR immunity that causes immunity amount and the animal that preferably can produce anti-CNTFR antibody and/or cell premunition of these models.In order to produce a kind of like this model system, just wish CNTFR with immunological adjuvant such as bacille Calmette-Guerin vaccine (BCG) administration.

5.6.2.1. the active model of enhanced CNTF

For example, but be not limited to, can prepare the experimental model system that is used to study the active effect of excessive CNTF.In such system, the CNTFR that accelerates by design on the cell of model system can make its response ratio to CNTF not have the cell of so design to increase.But preferably optionally on the cell of normal expression CNTFR, increase the quantity of CNTFR.

Cell can be by designing so that the quantity of its CNTF increases with the virus infection that has CNTFR gene of the present invention.In addition, the CNTFR gene can offer cell through transfection.

If model system is an animal, recombinant C NTFR gene can be by being incorporated in the cell of animal with the virus infection that has the CNTFR gene so.In addition, can obtain the CNTFR gene as genetically modified transgenic animal.

In order to guarantee the expression of CNTFR, the CNTFR gene should place under the control of suitable promoter sequence.Wish the CNTFR gene is placed under the control of composing type and/or tissue-specific promoter, promotor includes but not limited to that CNS neuron specific enolase, neurofilament and tyrosine hydrozylase promoter, a kind of inducible promoters are such as metallothioneine promotor, long terminal repetition UV activatory promotor (the Valeri et al. of people's HIVvirus blood serum immunity, 1988, Nature 333: 78-81) or CMV promotor (as containing among the pCMX hereinafter) or improve and regulate promotor.

The number of CNTFR by increasing cell, the response of internally giving birth to CNTF can be enhanced.If model system contains seldom or do not contain CNTF, so just can join CNTF in this system.Also wish other CNTF is joined in this model system so that improve the active effect of excessive CNTF.Undue CNTF(or the excretory CNTF of expressing) be that the method to the effect of improving the standard of the cell CNTF that expresses CNTFR is studied in preferable being used to.More preferably be in all cells, to express CNTFR(generally to express) and determine it is that those cells are that CNTF gives the sense response subsequently, thus may identify secondary acceptor component, if an existence is arranged.

5.6.2.2. the active model of CNTF that reduces

On the other hand, not as limiting as an example, can set up an experimental model system that is used to study the active effect of CNTF of reduction.This system can identify the method or the neurone of needed CNTF and the therapeutic goal that expresses possibility.In such system, can be to the response of CNTF by providing not relevant or being designed to reducing in response to the invalid recombinant C NTFR of the transduction of CNTF with cell surface.

For example, the acceptor that provides can offer this system to CNTFR protein, peptide or derivative so that can be connected with endogenous CNTFR competition CNTF, thereby weakened the response to CNTF, CNTFR can be a kind of acellular acceptor, it or be added in this system or produce by this system.For example, the CNTFR protein that lacks the transmembrane zone can be used intrasystem cells produce, can secretion come out from the cell that produces such as no anchor CNTFR.In addition, CNTFR protein, peptide or derivative can join in the intrasystem extracellular space.

In another embodiment of the present invention, recombinant C NTFR gene can be used for giving birth to gene in homologous recombination inactivation or " getting ", thereby produces a kind of CNTFR disappearance cell, tissue or animal.For example, but be not to limit, recombinant C NTFR gene can be designed to contain a kind of insertion for example suddenly change neo gene and inactivation CNTFR.A kind ofly like this be structured in the control of suitable promotor and use down such as technology such as transfection, transduction, injection and be introduced in the cell, such as embryonic stem cell.The cell that contains this structure then can be selected with the G418 resistance.The cell that lacks complete CNTFR gene then can be by Southern trace or Northern trace or the evaluation of for example expressing of mensuration.The cell that lacks complete CNTFR gene can be fused in the early embryo cell then to produce the transgenic animal of CNTFR disappearance.A kind of like this animal is compared with the animal of not expressing the interior CNTF of giving birth to and shows, perhaps two kinds of phenotypes are mated fully or not exclusively mated, and are meaning the existence of other class CNTF factor or acceptor.

A kind of like this animal can be used for defining concrete neurone population or any other live body process, generally depends on CNTF.Therefore, if thereby animal do not express CNTFR and can not respond CNTF, these populations or process just are hopeful to take place so.

In addition, can be expressed on the intrasystem cell surface, but can be designed to the response that to transduce CNTF is connected with recombinant C NTFR protein, peptide or the derivative of interior living receptor competition CNTF.

Living CNTFR was connected on the CNTF the avidity of the avidity of CNTF in above-mentioned recombinant C NTFR protein, peptide or derivative can be similar to or be different from.For more effectively reducing the response to CNTF, CNTFR protein, peptide or derivative are preferably with the avidity greater than the shown avidity that goes out of natural receptor and are connected on the CNTF.

If CNTFR protein, peptide or derivative produce in model system, CNTFR protein, peptide or the derivative of nucleic acid encoding just can offer this system by infection, transduction, transfection etc. or as transgenosis so.As indicated above, the CNTFR gene can place under the control of suitable promotor, and promotor can be for example tissue-specific promoter or inducible promoters or improve and regulate promotor.

In a specific embodiments of the present invention, the interior living CNTFR gene of cell can replace by the homologous recombination CNTFR gene that suddenlyd change.

In another embodiment of the present invention, CNTFR expresses and can reduce by the CNTFR express cell is provided, and this cell contains anti-RNA of meaning of CNTFR or the DNA that its content is enough to reduce the CNTFR protein expression.

5.6.3. diagnostic uses

According to the present invention, the CNTFR probe can be used for identifying the cell and the tissue that are in normally or have diseased state that can respond CNTF.The invention provides the method that is used to identify the cell that can respond CNTF, comprise the expression of detection CNTFR in these cells.CNTFR express can by CNTFR mRNA transcribe or the proteinic production of CNTFR confirms.CNTFR expresses to use and identifies that CNTFR nucleic acid or proteinic probe detect.

A kind of to can be used for detecting the probe that CNTFR expresses be nucleic acid probe, and it can be used for utilizing any method known in the art to detect the RNA of CNTFR coding, and these methods include but not limited to: in situ hybridization, Northern engram analysis or PCR correlation technique.

Another kind of spendable probe is the CNTF of mark, as being to describe in the U.S. Patent application of No.07/532 285 at application number, at this its full content is added herein as a reference.

According to the present invention, term " mark " but CNTF is meant the CNTF molecule that is connected on the 2nd detection compound (" mark ").But detection compound can comprise radio isotope, fluorophor or can be connected to ligand on the acceptor or material that available colorimetry detects or has catalytic activity.In preferable embodiment, mark can contain antigenic determinant so that antibody can be connected on the mark.In another embodiment, mark itself can be a kind of antibody; In a kind of specific embodiments of the present invention, be labeled as monoclonal antibody RP3-17.Wish that mark does not disturb the biological activity of CNTF and the detection method of mark not to disturb CNTF to be connected on its acceptor basically.

Mark can be linked on the CNTF with any methods known in the art.Preferable embodiment of the present invention is that mark is covalently bound to CNTF, but wishes that in some cases mark links to each other (for example, if mark contains immunoglobulin molecules) with the non covalent bond bonding force.

Mark can be any be suitable for keeping its telltale official can and do not change the bioactive molecular dimension of continuous CNTF basically.If mark is for antigenic determinant being provided, so just wishing that it contains 5-15 the amino acid of having an appointment at least.

Explanation rather than qualification as an example, a kind of preferable concrete grammar of the present invention is, CNTF can use " fragment " polymerase chain reaction to mark, and the human BDNF of wherein recombinating (CNTF) is designed to have ten amino acid corresponding to the known antigens stator at its C-terminal.For example, but be not that conduct limits, this antigenic determinant can be corresponding to the defined epitope of people c-fungi (c-myc) proto-oncogene protein matter.

For example, but be not as limiting, " fragment " PCR method is as described below to can be used for linking ten amino acid fungi marks (the invention provides with similar approach banded amino acid mark).5 ' PCR primer corresponding to the CNTF sequence that is arranged in specific limited enzyme division upstream, site in bacterial expression makes up can be used for reacting with the PCR of " fragment " primer, wherein use cDNA from the cell of CNTF response as template, said " fragment " primer contains peptide-labeled corresponding to the nucleotide sequence of 3 ' terminal CNTF sequence and nucleic acid sequence encoding.

The PCR reaction also contains corresponding to the fragment primer sequence and comprises 3 ' primer of the nucleotide sequence that contains specific limited restriction endonuclease division site.Preferable embodiment is, 5 ' and the consumption of 3 ' primer surpass the fragment primer, as a result 5 ' and the fragment primer between PCR be amplified in several PCR week after dates and just stop, and 5 ' and 3 ' primer between amplification can start and continue a large amount of total length CNTF/ flag sequence of production." fragment " technology can solve the needs to long primer, and the synthetic of long introduction is difficulty and consuming time.The restriction enzyme lixiviate can be purified, use to the CNTF/ marked product that amplifies through gel, and subclone is in the respective limits site of expression vector then, and wherein restriction enzyme divides on the site of the end that is designed into product.For example, in order to produce CNTF-fungi mark, can use following primer: 5 ' primer=5 ' GAC TCG AGT CGA CAT CGG AGG CTG ATG GGA TGCC 3 ' (SEQ ID No:3); Fragment primer=3 ' CTA AAG ACT CCT CCT AGA CAT CGC CGG CGT ATCG 5 ' (SEQ ID NO:4); The operable ratio of primer is 100ng5 ' introduction/100ng3 ' introduction/1ng fragment introduction; Detailed description see below 6 the joint.The expression of CNTF/ mark can be carried out the description of expression of reorganization CNTF in following file according to people such as Sendtner: the applying date is that August 20 nineteen ninety, application number are 07/,570 651 U.S. Patent application, the open No WO 91/04316 of PCT that its name is called " Ciliary Neurotrophic Factor " or published on April 4th, 1991.

The present invention also provides and has contained immunoglobulin molecules or its part, for example Fc of antibody molecule, F(ab) ₂Or F(ab) ' segmental mark.Mark should be connected on the CNTF and can be polyclone or monoclonal antibody.

According to the present invention, the CNTF of mark can promote CNTF to cultivate under the connection of said cell or the condition of adhering to cell.In most of the cases, this can realize under the type culture condition.For example, in preferable embodiment of the present invention, cell can be cultivated about 30 minutes in the presence of the CNTF of mark.If mark is an antibody molecule, be preferably so CNTF at first is connected on the cell and subsequently washed cell add anti-CNTF antibody labeling then to remove the ligand that does not connect.

In specific embodiments of the present invention, be positioned at the CNTF of the mark on the cell surface of CNTF response, hereinafter be called target cell, the available analysis of growing thickly (rosetting assay) detects, wherein can be connected to the cell cultures that telltale cell on the mark has the CNTF/ mark, they adhere on the CNTF/ mark of target cell as a result, and the telltale cell that connects has formed the clump of the shape of growing thickly at the cell peripheral that contains the CNTF-mark.These can upward measure with the standard microscope conceptions of technology with plate cultured cells (plated cell), perhaps, on the other hand, can separate growing thickly and non-growing thickly cell with Density Gradient Centrifugation.In a preferable specific embodiments of the present invention, target cell can gathered in the crops in such as the substratum of the RPM1 1640 that has 10% fetal bovine serum and 2mM glutamine under the concentration of about 200 cells/well and cultivate with plate in Duo Jing (for example 60 wells) culture dish such as neuronal cell, and sedimentary cell is at the 5%CO of humidity ₂Cultivate about 16 to 24 hours so that cell attachment at 37 ℃ in the incubator of atmosphere.Then, excessive cell culture medium is removed and cell can be cultivated about 30 minutes at the CNTF of room temperature with mark.Cell has calcium and magnesium with the PBS(that has added 1% bovine serum albumin(BSA) (BSA) then) wash and cultivated about 30 minutes with the antibody of the identification marking molecule of about 10 μ g/ml in room temperature then to remove the ligand that is not connected for several times.The antibody that cell does not connect with removal for several times with the PBS washing then.Then, target cell (containing the CNTF/ mark that is connected on the anti-tag antibody) in room temperature with the about 0.2%(V/V that is connected to the telltale cell of growing thickly on the anti-tag antibody (such as the telltale cell that contains rabbit-anti--mouse immuning ball protein)) suspension cultivation 1 hour.Plate washs and checks under phase microscope with PBS then grows thickly.For example, if anti-tag antibody is produced by mouse, the telltale cell can produce by anti-(mouse immuning ball protein) antibody coating red corpuscle (such as people O+ red corpuscle) of producing with another species so.The telltale cell can by according to the program of Albino etc. (1981, J.Exp.Med.154: the 0.01%CrCl that 1764-1778) in salt solution, dilutes ₃6H ₂O exist down with AIA (concentration greater than about 1 mg/ml under) the cultivation red corpuscle prepares.In addition, can use magnetic bead known in the art (magne-tic bead) or other method.

In another embodiment of the present invention, can use immunofluorescence technique to detect at the CNTF of the lip-deep mark of target cell, wherein be preferably the molecule that reacts with antibody and produce fluorescence directly or indirectly with mark.Fluorescence can be examined under a microscope, and fluorescence can be used for intensifying the segregation that the cell divide technology contains the cell of CNTF/ mark by fluorescence.A preferable specific embodiments of the present invention is described as with example, target cell can be in the analysis buffer reagent that contains CNTF/ mark (concentration excess) and sodium azide (0.05%) about 30 minutes of 4 ℃ of developments and resuspending.Cell then can be through washing three times down at 800 rev/mins in analyzing buffer reagent in centrifugal 5 minutes.Cell was cultivated about 30 minutes with anti-tag antibody at 4 ℃ under about 10 mcg/ml concentration then, by as the upper type washing, cultivated about 30 minutes at 4 ℃ of (biotinylated) anti-immunoglobulin and streptavidin-texas Red (Texas Red) conjugates then with biotinylization.Cell is washed then, resuspending, cover plate are cultivated (Coverslipped), checked with fluorescent microscopy then in sealing solution (mounting solution).

The present invention also provides the method for other formal notation such as the mark that produces look, catalytic label etc. of checking.The detection method of any concrete mark depends on from the necessary condition of mark generation signal, but those of ordinary skill in the art should be able to easily discern.

Operable another kind of probe is anti-CNTFR antibody.

According to the present invention, CNTFR protein or fragment or derivative can be used as immunogen to produce anti-CNTFR antibody.Can produce a large amount of CNTFR protein by the recombinant technology (based on CNTFR nucleotide sequence of the present invention) that uses protein synthesis, like this this problem of CNTFR with regard to having avoided limiting the quantity of.

For further improving the possibility of producing anti-CNTFR immune response, the aminoacid sequence of reply CNTFR is analyzed to identify the part of the molecule relevant with the immunogenicity that increases.For example, aminoacid sequence is carried out Computer Analysis to identify surface antigen decision base, and epitope has been represented the sub-district by the computer generation of the secondary structure of wetting ability, surperficial probability, flexibility ratio, antigenic index, amphiphilic spirane structure, amphiphilic layer and CNTFR.In addition, the aminoacid sequence of the CNTFR of the different plant species of derivation can compare, and relative non-homogeneous district is identified; These non-homogeneous districts more likely are to be immunogenic to various species.

In order to prepare the monoclonal antibody of pointing to CNTFR, any technology for preparing antibody molecule by the continuous cell line in substratum all can be used.For example, initial hybridoma technology (1975 by kohler and Milstein research, Nature 256: 495-497) and trioma technology, human B cell hybridoma technology (Kozbor et al., 1983, Immu-nology Today 4: 72) and EBV hybridoma technology (the Cole et al. that produces human monoclonal antibodies, 1985, in " Monoclonal Antibodies and Cancer Therapy ", Alan R.Liss Inc.pp.77-96) or the like all belongs to scope of the present invention.

The monoclonal antibody that is used for the treatment of can be human monoclonal antibodies or mosaic people-mouse (or other species) monoclonal antibody.Human monoclonal antibodies can be with many technology productions known in the art (Teng et al. for example, 1983, Proc.Natl.Acad.Sci.U.S.A.80:7308-7312; Kozbor et al., 1983, Immunology Today 4: 72-79; Olsson et al., 1982, Meth.Enzymol.92: 3-16).The chimera antibody of preparation can contain mouse antigen connecting zone and human constant region (Morrison et al., 1984, Proc.Natl.Acad.Sci.U.S.A.81: 6851, Takeda et al., 1985, Nature 314: 452).

Various techniques known in the art can be used for producing the polyclonal antibody of CNTFR epitope.For producing antibody, can make various host animal immunity through injection of CNTF R protein or fragment or derivatives thereof, said animal includes but not limited to: rabbit, mouse, rat etc.Various adjuvants can be used for the enhancing immunity response, this depends on host species, and adjuvant includes but not limited to: Freund adjuvant (completely with incomplete), mineral coagulant are such as aluminium hydroxide, surfactant segmented copolymer polyvalent alcohol (pluronic polyol), polyanion, peptide, oil emulsion, the key hole such as lysolecithin, epoxy ethane-epoxy propane

Hemocyanin, dinitrophenol(DNP) and people's adjuvant with potential use are such as the BCG(bacille Calmette-Guerin vaccine) and CBP (Corynebacterium parvum).

The molecular cloning of the antibody of CNTFR epitope can prepare with known technology.Recombinant DNA method is (referring to for example Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) can be used for making up the nucleotide sequence of coding monoclonal antibody molecule or its antigen joining region.

Antibody molecule can be used known technology, for example immunosorption or immunoaffinity chromatography, such as the HPLC(high speed liquid chromatography) chromatography or their combination etc. purify.

The invention provides the fragment of antibody molecule and these antibody molecules.The Id antibody fragment that contains molecule can be produced with known technology.For example, these fragments include but not limited to: F(ab ') ₂Fragment, the gastric pepsin digestion production of available antibodies molecule; Fab ' fragment can be through reducing F(ab ') ₂Segmental disulfide linkage is produced; And the Fab fragment, can produce through handling antibody molecule with papoid and reductive agent.

Above-mentioned probe can be used to identify the cell or tissue that also is not used to express CNTFR so far experimentally.In addition, these methods can be used to identify the expression of CNTFR by the distortion tissue such as cancer.In other embodiments, these method judgement ground are used for the expression of the cell that suffers disorderly patient, liquid or tissue CNTFR is compared to healthy people's similar cell, liquid or tissue.Liquid refers to liquid in any body, but is meant blood or celiolymph especially.Patient shows with the difference of healthy physiognomy than its CNTFR expression levels, patient's disorder mainly or inferior strategic point relevant with the CNTF metabolism.The increase of CNTFR level, for example, the disorder that perhaps shows patient is relevant with the susceptibility to the raising of the normal level of CNTF, perhaps on the other hand, shows that patient's CNTF level is low, and the number of acceptor is owing to compensation increases as a result.These cause of diseases can be separated from each other by allowing patient take CNTF.If patient's situation worsens, he just suffers from CNTF allergy; If patient takes a turn for the better, he just suffers from the CNTF disappearance.Thereby can select CNTF or based on the treatment plan of CNTF antagonist.The difference of expressing can detect under protein and/or rna level; Promptly by measuring patient's amount with respect to healthy people's CNTFR protein or CNTFR RNA.

Above-mentioned probe also can be used for selecting being used for the cell of the CNTF response of mensuration system, as mentioned above, perhaps is the U.S. Patent application of No.07/532 285 referring to application number, perhaps selects according to cell or the standard method of cell divide.

5.6.4. therepic use

The present invention also provides the CNTFR protein, peptide fragment or the derivatives for treatment that use significant quantity of the present invention to suffer from the method for disorder as the patient of nervous disorders.Comprise with CNTFR, CNTFR stimulant, CNTFR antagonist (it and interior living CNTF competition) or the methods of treatment of anti-CNTFR antibody administration and all belong to scope of the present invention.

The present invention also provides the pharmaceutical composition that contains CNTFR protein, peptide fragment or derivative in suitable pharmaceutical carrier.

CNTFR protein, peptide fragment or derivative can be capapie or administration partly.Any suitable administering mode known in the art all can use, and includes but not limited to: in the intravenously, canalis spinalis, interior, oral, subcutaneous, the intraperitoneal of intra-arterial, nose or local injection or surgery implantation.The persistence release formulation also is provided.

When we were familiar with to such an extent that more know to neurodegenerative disease/neural wound, the benefit that reduces endogenous CNTF trophism was just more obvious.So in the nervous system trauma zone, Chang Wang provides CNTF antagonist, comprise (but being not limited thereto) acellular CNTFR, this CNTFR can combine with endogenous cell acceptor contention CNTF.In these cases, provide CNTF to meet the requirements at damage location rather than in the whole body part.It may be desirable that use provides the implant of CNTFR.

In addition, some pathological state may obtain an advantage from the raising of CNTF responsiveness.Therefore, in suffering the patient of this pathological state, increase the quantity of CNTFR or may be useful in conjunction with affinity.This can reach by gene therapy.Use is by the CNTFR gene of tissue specificity or evoked promoter control, or by with have recombinant chou CNTFR gene duplicate defective virus produce selection that local infection obtains the recombinant chou CNTFR in suitable cell express those can benefit from increase to the pathological state of CNTF susceptibility particularly including the disorder of (but being not limited to) motor neuron, comprise amyotrophic side sclerosis and back poliomyelitis (post-polio) syndromes.This therapy also can be used for treating and diabetes, Parkinson's disease, the Al-zheimer disease nervous disorders relevant with the Huntington tarantism.

In addition, the present invention provides treatment to particular tissues or cell type disorder by the CNTF administration, and this class tissue or cell type identify as expressing the CNTF acceptor.In specific embodiments, shown this CNTFR gene quilt expression in muscle cell (the 8th joint sees below), and this CNTF can prevent the loss (the 9th joint sees below) of muscle weight and the fribrillin content relevant with denervation atrophy in the body.Therefore, the invention provides the disorder of treatment muscle cell, or comprise the method for neuromuscular unit disorder, this method comprises that the patient to this treatment of need takes: (ⅰ) nucleic acid molecule or the functional activity part or derivatives thereof of the nucleotide sequence of the CNTFR that encodes contained at a place, so that it can be expressed, or (ⅱ) CNTF, or its functional activity part or derivatives thereof.This disorder includes but not limited to amyotrophy or malnutritive variation that basic pathology is found.For example, this amyotrophy may be by the denervation that causes because of neural wound (muscle loses with its nerve and contacts); Degenerate (for example Ge-Ba Er Shi (Guillian-Barre) syndromes) peripheral neurophaty of metabolic or inflammation or cause by environmental poisonous substance or drug-induced damage to nerve.In another embodiment, amyotrophy is to be caused by the denervation that causes because of motorius.This motor neuron includes but not limited to the motor neurone disease of being grown up, and comprises amyotrophic side sclerosis (ALS or LouGehrig ' s disease); Infant's vertebra amyotrophy and have the autoimmune motor neuron of many focuses conducting region.In another embodiment, amyotrophy is caused with property by chronic giving up.This class disuse atrophy can be produced by following these pathological states, include but not limited to: since in wind-induced paralysis, Spinal injury, brain injury or other central nervous system injurys are because fixation of skeleton or CBR that wound (as fracturing, sprain or misplacing) causes.In one embodiment, amyotrophy is by metabolism Stress or malnutritive causing, this includes but not limited to the emaciation of cancer and other chronic diseases, jejunitas or rhabdomyolysis (rhabdomyolysis), and endocrine disturbance is as imbalance of (but being not limited to) Tiroidina and diabetes.Amyotrophy is also because the muscular dystrophy syndromes causes the malnutrition (ocu-lopharyngeal), omoplate humerus, limb girdle and congenital type and that be known as hereditary distally myopathy (Hereditary Distal Myopathy) of this include but not limited to shut out Xing Shi (Duchenne), Bake Er Shi (Becker), myotonic, manadesma omoplate humerus (Fascioscapulohumeral), Emery-Dreifuss, eye pharynx.In another embodiment, amyotrophy is caused that by congenital myonosus this includes, but are not limited to optimum congenital hypotony (Benign Congenital Hypotonia), centronucleus (Central Core) disease.Nematode myopathy (Nemaline Myopathy) and tubulose flesh (Myotu-bular) (centronucleus) disease.In addition, CNTFR coding nucleic acid or CNTF and active fragments thereof or derivative can be used for treating the day after tomorrow (poisoning or inflammation) myopathy.Those are because the myopathy that the muscle inflammatory disease causes includes but not limited to polymyositis and dermatitis.Toxic myopathy can be caused by following these reagent, include but not limited to atlansil, chloroquine, clofibrate, Zorubicin, ethanol, Plaquenil, organophosphate, perhexiline and vincristine(VCR).

In other embodiment of the present invention, obtain medical treatment for the expression that suffers excessive CNTFR, can reduce CNTFR thus by sense-rna or the few deoxyribonucleotide of antisense of taking significant quantity corresponding to the CNTFR gene coding region to patients hypersensitive such as CNTF, excessive CNTF.

6. embodiment: the cloning by expression of the ciliary neurotrophic factor receptor

6.1. material and method

6.1.1.CNTF the structure in expression of receptor storehouse

SH-SY5Y cell (being obtained at first by Dr.June Biedler) is as the mRNA source in construction cDNA storehouse, and the pCMX expression vector (be disclosed in await the reply simultaneously March in 1991 application serial number No.07/678408 on the 28th U.S. Patent application in, see above), this is a kind of derivative (Seed of pCDM8 carrier, 1987, Nature 329: 840-842).Select to be used for the inset in cDNA storehouse on sepharose, its chi is crossed greater than 1kb.

" 6.1.2. pan analysis (panning) " method

To " pan analysis " method (1987 by Seed and Aruffo exploitation, Proc.Natl.Acad.Sci.U.S.A.84: 3365-3369) make the following changes: cell at first uses CNTF/ fungi (1 μ g/ml) to cultivate 30 minutes on ice, rotation is by the part of PBS/2%Ficoll to remove, use 9E10 antibody (by Oncogene Sciences then, Manhasset, N.Y. obtain) cultivated 30 minutes on ice, rather than come culturing cell with the antibody of identification receptor.Again PBS/2% Ficoll is passed through in its rotation subsequently, and on the plate that is covered with the anti-fungus peptide mouse monoclonal antibody that obtains from Sigma company " pan analysis ".Being prepared as follows of this plate: will be covered with 60mm plate (Falcon 100) or its equivalent of antimycotic mouse monoclonal antibody bacterium, or the 10cm ware) be diluted to every milliliter 10 microgram as Fisher 8-757-12, with 50mM Tris HCl(pH9.5.The antibody of 3ml is used to cover each 6cm ware or 10ml is used for each 10cm ware; Plate was exposed to antibody about 1.5 hours, then antibody is transferred in next ware, make to keep 1.5 hours, and then transfer in the 3rd ware.Wash this plate three times (being more convenient with washing bottle) with 0.15M NaCl for this reason, in PBS, use 3ml 1mg/ml BSA overnight incubation then.Especially by following carrying out " pan analysis ": cell is cultivated in the 100mm ware.Sucking-off substratum from each ware adds 2ml PBS/0.5mM EDTA/0.02% trinitride again, and this mixture is cultivated 30 minutes down so that cell is separated from ware at 37 ℃.Firmly these cells are smashed to pieces with short Pasteur (pas-teur) suction pipe, from each ware, be collected in the centrifuge tube, and 2.5 positions (200xg) rotation 4 minutes.Cell suspension in 0.5-1.0ml PBS/EDTA/ trinitride/5%FBS, and was cultivated on ice 30 minutes with the CNTF/ fungi.Add isopyknic PBS/EDTA/ trinitride, careful layering in 3ml PBS/EDTA/ trinitride/2% Ficoll, the rotation of 2.5 places is 4 minutes in the position, with supernatant liquor sucking-off reposefully.Use then 9E10 antibody on ice with these cell cultures 30 minutes, heavily cover rotation again by PBS/EDTA/ trinitride/2%Ficoll.These cytolysises in 0.5ml PBS/EDTA/ trinitride, are joined aliquots containig on the ware of the antimycotic mouse monoclonal antibody covering of the usefulness that contains 3ml PBS/EDTA/ trinitride/5%FBS.From 2 60mm wares, cell is joined in 1 antibody-coated plate of 60mm at the most, and make room temperature placement 1-3 hour.By washing lentamente and will not adhering to excessive cell on the ware and remove (usually with 3ml washing 2-3 time just enough) with PBS/5% serum or with substratum.

6.1.3. contain the evaluation of the ciliary neurotrophic factor receptor gene clone

Will be according to standard method use DEAE/ chloroquine from plasmid DNA transfection (about 250-500ng of every 100mm ware in the COSM5 cell of expression library; Two wares of transfection).After the transfection two days, cell is broken away from from its ware, and stand the pan step of improved Aruffo/Seed as indicated above.

Behind plate washing non-adherent cell, and preparation Hirt supernatant liquor (Hirt, 1967, J.Mol.Biol.26: 365-369), precipitation plasmid DNA when having 10-20 μ g tRNA.According to the explanation of manufacturer by electroporation the DNA that obtains is introduced a fine variety in the DH10B bacterium (Electromax, BRL).Substratum by the bacterial growth of electroporation is used to prepare transfection and the pan of plasmid DNA to be used for next round; By indirect iodate-antibodies test, obviously show as the COS cell (see the representative data of Figure 1B, measuring method sees below) of a large amount of expression CNTFR with a plate COS cell of this plasmid DNA transfection.Carry out on these transfectants second take turns pan/plasmid DNA separation/electroporation after, turn out the bacterium transformant that obtains by the electroporation step at penbritin plate upper flat plate.Collect individual bacterial clone, by each plasmid DNA that should clone prepares individually transfection in the COS cell to be used for mensuration.15 14 of testing in the plasmid by a series of mensuration, comprise that indirect antibodies as mentioned below is measured and fluorescence amplifying cell separator (FACS) is analyzed, and the result forms and expresses the CNTF binding site in the rotaring redyeing COS cell.

6.1.4. directly ¹²⁵I-hCNTF is in conjunction with mensuration

Use from the storehouse, strengthen storehouse or individual clone's plasmid DNA transfection COS cell.After 48 hours, remove substratum, and with only containing ¹²⁵The 0.25ml binding buffer liquid of I-hCNTF (has 10%FBS and 0.1%NaN ₃RPM1 1640) or replace with unlabelled hCNTF.Use in room temperature ¹²⁵I-hCNTF cultivated 60 minutes.After cultivating fully, remove this ¹²⁵I-hCNTF solution, and wash these cells three times with 1.0ml binding buffer liquid is then with the 0.1N NaOH dissolving of 0.25ml.Transfer to lysate in 12 * 75mm polystyrene tube and place the r counter.Measure non-specific binding by adding at least 100 times of excessive unmarked hCNTF.After final washing, this plate is carried out radioautogram.

6.1.5. fluorescence amplifying cell separator analysis

The COS cell of transfection is cultivated with the anti-mouse antibodies of CNTF/ fungi, 9E10 antibody and FITC-labelled goat successively.Break away from and the process facs analysis from ware then.Result with the negative, positive plasmid transfection is plotted among Fig. 1 D; COS cell with CNTF expression of receptor plasmid transfection contains a large amount of subgroups, demonstrates fluorescence by this mensuration and strengthens greatly.

6.1.6 the iodate of hCNTF

Use lactoperoxidase 6ng/ μ l(Sigma at 20 ℃) use 1mci ¹²⁵INa sulfonation 10 μ g hCNTF(560 μ g/ml are at 10mM NaPO ₄In, pH7.4) 15 minutes.After 15 minutes, with containing 0.1M NaI, 0.1%BSA and 0.1% cytochrome C .0.3%HOAC, 0.05% phenol red and 0.02%NaN ₃Equal-volume damping fluid inhibited reaction.Shift out aliquots containig to be used to measure the precipitation quantity of TCA.Remainder installed to be loaded with 0.05M NaPO ₄, 0.1M NaCl, 0.5mg/ml protamine sulfate and 1mg/ml BSA BioRad PD-10 xanthan gel post on.Collect cut and measure TCA precipitation number.

6.1.7 the sequential analysis of CNTFR

According to the explanation of manufacturer, use dideoxy two-wire DNA element (sale of U.S.Biochemi-cal company) to use Sequenase ^TMCarry out sequential analysis

6.1.8. it is indirect ¹²⁵The I goat anti-mouse antibody is in conjunction with mensuration

Use from the storehouse, strengthen storehouse or individual clone's plasmid DNA transfection COS cell.After 48 hours, contain Ca, Mg with PBS(successively on ice)/5%FBS cultivated these cells 30 minutes, and 5%FBS contains:

1) 1 μ g/ml CNTF-fungi;

2）10μg/ml9E10;

3) ¹²⁵I goat anti-mouse antibody (GaM) (0.5-1 μ Ci/ml).

After each step, cell was washed in PBS/5%FBS 3 * 5 minutes.After the last washing, this plate is carried out radioautogram.

For the individuality clone, measure its bonded gross activity quantitatively with portable r counter.

6.2. result's discussion

6.2.1. restriction analysis

When restriction analysis, 14 just cloned be divided into 4 groups:

a）I2＝I7（2kb）

b）I1＝I5＝I6（2kb）

c）I4＝I8＝I9＝I11＝I14＝I15（4kb）

d）I10＝I12＝I13（1.6kb）

(I3 is negative)

When digesting with enzyme Pst I, the member of each group produces identical wounded in the battle sample.Further restriction analysis shows that four groups of the clone is eclipsed, the digital proof of initiation sequence, and they are in the total eclipsed sequence of its 5 ' end.Curiously, its nuclear promoter element is organized b relatively) show that its inset is arranged on the misorientation of carrier.As table 1 as seen, with respect to other clones, these clones' expression is low.Transcribing in these clones can produce by there is weak hiding promotor in the catchment of the many linkers of carrier.

6.2.2. in-vitro transcription and translating

For characterizing protein coding, they are all transcribed by the T7 promotor in 5 of the many linkers of carrier ' district four groups of clones.After external translating, product is carried out electroporation on polyacrylamide gel.Group a) does not produce protein, and this is because be positioned at wrong orientation with respect to the T7 promotor.Other the three groups protein (about 42kd) that all produce same size, this proves the identical protein of they codings.

The table I

In the CNTFR clone ¹²⁵I-GaM bonded quantity

The clone is in conjunction with CPM

Ⅰ1 2000

Ⅰ2 8500

Ⅰ3 600

Ⅰ4 9000

Ⅰ5 2000

Ⅰ6 1600

Ⅰ7 6000

Ⅰ8 7500

Ⅰ9 7000

Ⅰ10 4500

Ⅰ11 7000

Ⅰ12 5000

Ⅰ13 8000

Ⅰ14 10000

Ⅰ15 8000

Negative contrast 500

Background 250

6.2.3. binding analysis with CNTF

Use the 9E10 anti fungal antibody and ¹²⁵The indirect CNTF-fungi of I goat anti-mouse antibody is in conjunction with the results are shown in Figure 1B and 1C and the table 1 of measuring.In Figure 1B, the plate of on the left is obtained by the transfection that does not strengthen the storehouse, and the plate on the right is from (the using and the DNA that does not strengthen the about equal amts in storehouse) of carrying out taking turns the enhancing storehouse plasmid DNA transfection that obtains behind the pan.Only observe a large amount of stains in the plate on the right, the single COS cell expressing CNTF-fungi binding site that on behalf of radioautograph, each stain all detect.

Just cloning for the individuality of in the 6.2.1. joint, discussing, carrying out the quantitative assay of gross activity with portable r counter.The measurement result of individuality clone I1-I15 is shown in Table 1 and shows that 14 among 15 clones are expressed CNTF binding sites (recording by indirect ABT).In addition, shown in Fig. 1 C, the fragment in the plate of cloning from some individuality is carried out radioautogram.

Shown in Fig. 1 D, the CNTF-fungi has been used in the bonded experiment indirectly for the second time, uses 9E10 antibody, the anti-mouse antibodies of FITC labelled goat subsequently, and facs analysis.With the COS cell of just cloning transfection with compare with negative clone's cells transfected, what show that it improves CNTFR is expressed as 100 times.

Direct with the indirect binding data that the CNTF-fungi obtains by using ¹²⁵I-CNTF the results are shown in the table II in conjunction with verifying.This receptor of expressing on the COS of transfection cell especially easily combines with iodinating CNTF and CNTF-fungi part as the SH-SY5Y cell that CNTFR is cloned.The COS cell expressing of each transfection is than the high about 30 times of every cell receptors of SH-SY5Y cell.

The table II

With iodate the CNTF binding analysis

COS Ⅰ2 SH-SY5Y

¹²⁵The cpm/ cell that I-CNTF is special ^*Special cpm/ cell

Concentration in conjunction with cpm in conjunction with cpm

2.16nM 1412 2.17×10 ^-21284 4.28×10 ^-3

In 24 well culture dish with 3 * 10 ⁵Individual SH-SY5Y cell/well or 6.5 * 10 ⁴Individual COS cell/well carries out single layer and measures.Special calculating in conjunction with cpm is from only with specified concentration ¹²⁵During I-CNTF in conjunction with deduct among the cmp when having 1000 times of excessive unlabelled CNTF in conjunction with cpm.In the COS of untransfected cell, do not detect special combination.

^*By with DEAE Dextran(diethylaminoethyl dextran) carried out the COS raji cell assay Raji after the transfection in 48 hours, a transfection 20-40% therein typically.Suppose the COS cell of transfection 20%, the special COS cell every cell receptor higher approximately 30 times that shows each transfection in conjunction with cpm than SH-SY5Y cell expressing.

6.2.4.CNTFR with coming from other growth factor receptors sequences

This CNTFR contains the theme total with a series of other acceptors.Born of the same parents' outside part of CNTFR contains and is positioned at " immunoglobulin (Ig) " zone that N-does not hold, and by short zone, twisting district isolated from " immunoglobulin (Ig) " " cytokine acceptor ".Although a lot of acceptors not only with " immunoglobulin (Ig) " but also with " cytokine acceptor " homology (Fig. 3), having only an acceptor is the identical particular arrangement (Fig. 3 and Fig. 4) that IL-6 acceptor and CNTFR have these zones.So this IL-6 acceptor is and the maximally related protein of CNTFR (Fig. 4).Meaningfully, this IL-6 acceptor also is similar to CNTFR, and it has very short kytoplasm zone therein, obviously for IL-6 in conjunction with do not need initial response (Hibi et al., 1990, Cell 63: 1149-1157).Recently, a kind of new signal transducer to the IL6 acceptor is named and is to have carried out molecular cloning by gp130.The own debond IL-6 of this transducer, but it provides the high affinity that is attached on the IL-6 acceptor, and for transduction IL-6 signal required (Hibi et al., 1990, Cell 63: 1149-1157).We show that to the clone of CNTFR it and IL-6 acceptor have important feature, and this is not find in known other acceptors, so, defined a new acceptor family.Similarity between IL-6R and the CNTFR shows that CNTFR can use identical signal transducer as the IL-6 acceptor, or relevant molecule.At last, the evaluation of the acceptor that CNTFR is relevant can by means of with the evaluation of the new part of these receptors bind.

7. embodiment: the tissue to CNTFR information is surveyed

7.1. the method for material

7.1.1.CNTFR the preparation of probe

The coding region molecular cloning of hCNTFR is gone in the pCMX expression vector, and this exercise question that is disclosed in application meanwhile is in the U.S. Patent application of " mammiferous expression vector ", and resulting expression vector is plotted among Fig. 6.Synthesized the PCR probe that extends to base pair 1230 by CNTFR sequence base pair 889, and it has been used as the Northern analysis probe.

7.1.2.RNA preparation and Northern trace

From the Sprague-Dawley rat, separate and cut selected tissue and freezing immediately in liquid nitrogen.As described in the people such as Bothwell (1990, Methods of Cloning and Analysis of Eukaryotic Genes, Boston, MS, Jones and Bartlett) by the homogenizing of in 3M LiCl, 6M urea, organizing RNA is isolated.By through the electrophoretic method of four 1% agarose-formaldehyde gel with RNA(10 μ g) particle (Bothwell et al., 1990, Methods of Cloning and Analysis of Eukaryotic Genes, Boston, MS, Jones and Bartlett), then with capillary transfer to having on 10 * SSC(pH7) the nylon membrane (MagnaGrath, Micron Separation Inc.).(Stratalinker, Stratagen Inc.), and are having 0.5M NaPO under 68 ℃ to this film with the RNA ultraviolet-crosslinkable under the ultraviolet light by being exposed to ₄(pH7), 1% bovine serum albumin (cut V, Sigma, Inc.), 7%SDS, 1mM EDTA(Mahmoudi et al., 1989, Biotechniques 7: 331-333), during the sex change salmon sperm DNA of 100 μ g/ml sonications and radiolabeled probe hybridization.68 ℃ with 3 * SSC, 0.1%SDS washing nozzle and 70 ℃ down with one or two reinforcing membrane (Cronex, Dupont) and x-ray film (SAR-5 Kodak) carries out radioautogram one day to two weeks.To the bromination 3 of gel, drone the dyeing of 8-diamino-5-ethyl-6-phenylphenanthridineand show the level of total RNA that various sample is measured be suitable (as Maisonpierre et al., 1990, Science 247: 1446-1451).

7.2. result

As shown in Figure 5, in the central nervous system tissue when CNTFR mRNA is low-level in sciatic nerve and suprarenal gland, and be detectable in muscle.This shows that CNTF not only has neurotrophic activity, and has a muscle nutrition activity, and can illustrate the central nervous system that relates in particular disorder unify muscle both, these disorders for example shut out Xing Shi muscular dystrophy and congenital myotonic dystrophy, in these diseases, patient suffers the spirit retardance.CNTFR expresses this explanation CNTF and may have certain effect by tool in muscle physiology in muscle.So except acting on nerve, CNTF may have important effect in muscle,, or in other words influence muscle development and/or differentiation as effect as the myotrophy agent.

8. embodiment: prove that the CNTF acceptor is connected on the cell surface by glycosyl-phosphatidyl inositol (GPI) key

8.1 material and method

In the RPMI that is supplemented with 10% non-activated fetal bovine serum, in 24 well wares (Falcon), cultivate the SH-SY5Y cell.In experiment, carrying out Phospholipid hydrolase (and contrast) before the CNTF combination handles, with the substratum sucking-off, use PBS(+Ca/Mg) cell is washed secondary, phosphatidylinositols-characteristic phosphide enzyme (PI-PLC) ultimate density is that 500mU/ml(purchases the product in the #1143-069 of Boehringer Mannheim with being supplemented with or not replenishing then) PBS(+Ca/mg) cultivated 45 minutes down at 37 ℃.Use binding buffer liquid (PBS(+Ca/Mg) and 5% fetal bovine serum then) with cell washing three times, and then contain about 100 pmols of iodinating CNTF() with 250ml, this CNTF contained or does not contain 1000 times of excessive unlabelled CNTF binding buffer liquid, incubated at room temperature 30 minutes.In experiment, before handling, PI-PLC, under 37 ℃, containing iodinating CNTF with iodinating CNTF combination, and this CNTF contains or does not contain in the binding buffer liquid of excessive unlabelled CNTF and at first cultivated 45 minutes.Use PBS(+Ca/Mg then) with the cell washing secondary, be 500mu/ml with replenishing or not replenishing the PI-PLC(ultimate density again) PBS(+Ca/Mg) cultivated 45 minutes.Then wash cell three times with binding buffer liquid.In all cases, dissolved cell before the counting in 0.1N NaOH, and then counting.

8.2. result and discussion

The sequence of CNTF acceptor shows that coded protein terminates in its heel and has in the hydrophobic region in outer kytoplasm zone, any tangible stop-transfer sequence or endoplasm zone do not occur.This structure make the people remembered do not have a transmembrane zone and be connected the C-end of finding on the membrane protein on the cell surface (Ferguson and Williams, 1988) by the GPI key.So whether experimentize to test this CNTF acceptor is to be connected on the cell surface by the GPI key.As show shown in the III, eliminated SH-SY5Y fully thereafter in conjunction with the ability of CNTF with PI-PLC treatment S H-SY5Y cell, this is consistent by the viewpoint that the GPI key is connected on the cell surface with this CNTF acceptor.Yet, can handle making the CNTF that is connected on the SH-SY5Y cell discharge (table III) by PI-PLC.Meaningfully, the IL-6 acceptor of soluble form can with required second membrane protein (GP130) of IL-6 signal transduction is closely combined.So, prevent on the CNTF that by being combined in advance the CNTF acceptor from discharging may be owing to bonded result between CNTF, its acceptor and signal transducer (GP130 or GP130 analogue) thereof.On the other hand, CNTF makes it to PI-PLC more insensitive (protein that some GPI connects has PI-PLC resistance form) in conjunction with the structure that can change the CNTF acceptor.

The CNTF acceptor is connected by the GPI key that this discovery has important result on the cell surface.This has represented to find for the first time that growth factor receptors is connected on the film by this way, has increased the possibility that additional acceptor can be connected by GPI.Because some protein have the GPI type of attachment and contain these two kinds of forms of common transmembrane area format, our discovery has increased the CNTF acceptor and has had the alternately possibility of the C-terminal in codified transmembrane zone, and is similar to the form that the IL-6 acceptor has the GPI connection.The GPI type of attachment of growth factor receptors can use new acceptor to regulate and releasing mechanism.For example, the downward adjusting of surface receptor can make the release of the acceptor that GPI connects by the phospholipase activity that activates outer kytoplasm and take place rapidly.The acceptor of these releases also can act on other cell (or independent or with CNTF combine in roughly the same mode), and soluble IL-6 acceptor shows in conjunction with IL-6 and activated the cell of expressing GP130.

Using PI-PLC that the CNTF acceptor is discharged this possibility that can block the CNTF effect has great importance.This can be used for verifying that the effect of observed CNTF is because the result of clone's CNTF acceptor.In treatment, when thinking that the CNTF activity is deleterious, PI-PLC can be used for discharging the CNTF acceptor, and may block the effect of CNTF.

If it is that this feature of this receptor just can be used for definition and this transduction molecule of molecular cloning so owing to formed quarternary complex between CNTF, its acceptor and proteinic signal transducer matter that the PI-PLC that the CNTF of CNTF acceptor can block discharges.

The table III

To being incorporated into CNTF's on the SH-SY5Y cell

The analysis that PI-PLC handles

In conjunction with cpm

The no cold cold excessive thing of excessive thing

Give processing with PI-PLC

No PI-PLC 1,440 370

PI-PLC 420 310 is arranged

PI-PLC is before in conjunction with CNTF

No PI-PLC 1,250 310

PI-PLC 1,060 300 is arranged

9.CNTF in external effect to the denervation rat skeletal muscle

Experiment purpose described herein is whether can prevent and the atrophy of denervation that at external effect and definite CNTF to denervation skeletal muscle for example muscle weight some phenotype relevant with the myofibrillar protein loss changes for the recombinant chou CNTF that checks purifying.We find that this CNTF acceptor comprises in tubulose flesh and the sarcoplast at skeletal muscle and express, and this CNTF can prevent the loss of muscle weight and the myofibrillar protein content relevant with the atrophy of denervation.

9.1.CNTF acceptor comprises expression in tubulose flesh and the sarcoplast at skeletal muscle

As shown in Figure 5, carry out the Nor-thern engram analysis on the RNA sample that in by multiple rat tissue, obtains to identify the main cellular targets of CNTF.Probe from human body CNTF acceptor coding region has been identified the 2kb transcription, the expression of these transcriptions is only limited to central nervous system usually, have only and unexpectedly in skeletal muscle, found its high-caliber expression, and in suprarenal gland and sciatic nerve, be found to be low expression level.

The particularly more detailed analysis of CNTF expression of receptor in skeletal muscle is by using Northern trace by the mRNA of the Skeletal Muscle Cell system preparation in human muscle's tubulose myocyte of special rat muscle type, purifying and some mouse and rat source by above carrying out (Fig. 7).When the human body probe is used for the CNTF acceptor, two kinds of mRNA kinds (2.0 and 1.7kb) in some muscle RNA samples, have been measured.Fig. 7 shows that the CNTF acceptor is all to express among both at the tubulose flesh in mouse (row 1 and 2) or rat (row 3 and 4) source with becoming the flesh muscle cell, and in ballism soleus muscle and the white expression (being respectively row 5 and 6) in the musculus extensor digitorum longus pedis (EDL) of quivering fast slowly of the redness of rat.This level that shows the CNTF receptor mrna all increases in soleus muscle (row 12) and EDL muscle (row 14), and these muscle are denervation for the first time 72 hours with respect to the contrast (being respectively row 11 and row 13) of its sham-operation offside.Meaningfully, the highest level of expression is observed in the RNA sample of the tubulose flesh that comes free human fetal skeletal muscle to obtain.These tubulose fleshes are cultivated, then before RNA separates with the fluorescence amplifying cell separator purifying to separate (row 8) with other nonmuscle cellses with inoblast.We notice and identified two kinds of different CNTF receptor mrna species on muscle cell Northern traces, and notice that 1.7kb CNTF acceptor information priority is C2C12mb(capable 1 sarcoplast) in express, this may represent a kind of alternately type of attachment of acceptor.

9.2.CNTF prevent the loss of muscle weight and the myofibrillar protein content relevant with the denervation atrophy

9.2.1. neureotomy surgical operation

The different animal groups that is used for these researchs is shown in following table IV.Comprise three animals in common one group.For all experimental group, cause the initial otch of about 20cm by middle leg (midthigh) the position skin of right hind.After this surgical procedure, the otch of 20cm is also cut to carry out sham-operation in the shank position in left hind.

In animal groups 2-6, the flatfish muscle of right hind is by carrying out denervation at the sciatic 2-5mm nodal plate of the surgical removal at middle leg position, to separate 32 to 35mm distally neural residul end (using the A mark in Fig. 8).The flatfish muscle in left side wherein carries out sham-operation by the innervation point 32 to 35mm that lentamente sciatic nerve is pulled away from flatfish muscle in contrast on this muscle.Anaesthetize (0.3g/Kg) following time when animal at slight Sodital, carry out all surgical operations.Animal groups 1(contrast) not making any neurectomy does not do any injection yet.All animal body weight averages are between 100 and 150 grams.

9.2.2. handle

The animal of group in 1 and 2 do not handled, and the animal in the group 3 is with containing 1mg/ml BSA(PBS/BSA) phosphate buffer saline (PBS) muscle (IM) injection, once a day, injected altogether 4 days.Muscle in the middle shank position of animal both sides all carries out multiple injection.The animal of group in 4 is with containing 1mg/ml BSA(CNTF/BSA) recombinant chou rat CNTF(1mg/Kg) the IM injection, once a day, totally 4 days.All carry out multiple injection in the both sides of animal as mentioned above.Animal in the group 5 is also injected with rCNTF/BSA every day, but by subcutaneous rather than IM injection.Animal in the group 6 is injected (SC) with PBS/BSA every day.

The table IV

The group # animal surgery operation plan neurectomy time handles

13 do not have 96 hours does not have

23 R-Den/L-Sham did not have in 96 hours

96 hours PBS/BSA(1mg/ml of 33 R-Den/L-Sham)

96 hours CNTF/BSA(1mg/Kg of 43 R-Den/L-Sham) (IM)

96 hours CNTF/BSA(1mg/kg of 53 R-Den/L-Sham) (SC)

96 hours PBS/BSA(SC of 63 R-Den/L-Sham)

The R-Den=right hind art that denervates; The sham-operation of L-Sham=left hind

9.2.3. muscle weight and protein analysis

The neureotomy surgical operation is killed animal by detruncation after carrying out 96 hours, and with flatfish muscle carefully by tendon to tenotomize.Soleus muscle is placed on ice the boat of weighing, removes tendon, muscle is weighed to prevent any drying immediately then with scalper.Be the homogenate of preparation fribrillin, collect the flatfish muscle that cuts off and in the cold house on ice with its rubbing, containing 0.32M sucrose and 3mM MgCl then ₂Stir evenly among the PBS (2.5%w/v).With this homogenate with about 800xg centrifugation, the use Bio-Rad Dye Binding(dyestuff of recommending according to manufacturer in conjunction with) method, supernatant liquor is measured the total myofibrillar protein of every muscle.

Fig. 9 shows that the flatfish muscle of denervation obviously reduces (P＜0.01) about 25% at 96 hours its net weight.The PBS/BSA injection of every day is to depending on the no effect of muscle weight loss of denervation.Yet, take from and use CNTF(1mg/Kg every day)/weight low about 5% of its offside sham-operation contrast of weight ratio of the denervation soleus muscle of the rat of BSA injection.The nt wt net weight of the flatfish muscle of the denervated and sham-operation that CNTF handles is significantly different with the weight of the contrast of not performing the operation.When every day injection (SC) totally 4 days (group 5), the denervation inductive loss that CNTF also shows myofibrillar protein has the effect of significantly preventing, and the reduction of proteinic loss and muscle net weight suitable (table V).

The table V

CNTF is to the effect of denervated soleus muscle protein content

Total sarcostyle

Muscle sample protein (mg/ muscle) %(vacation)

Group 1-is denervation 7.5 not

Group 2-denervation-do not inject 5.8 80

-false-do not inject 7.2

Group 3-denervation+PBS(IM) 5.2 75

-vacation-PBS(IM) 6.9

Group 4-denervation+CNTF(IM) 6.5 83

-vacation+CNTF(IM) 7.8

Group 5-denervation+CNTF(SC) 6.6 93

-vacation+CNTF(SC) 7.1

Group 6-denervation+PBS(SC) 5.3 67

-vacation+PBS(SC) 7.9

(IM)=intramuscular injection; (SC)=subcutaneous injection; The total protein content of 3 parts of soleus muscle of data represented collection in the table

When every day, IM injected, observe CNTF to the not significant effect of total myofibrillar protein.

We find that the CNTF acceptor comprises in tubulose myocyte and the sarcoplast at skeletal muscle and express, and CNTF can prevent the two the loss of the muscle weight relevant with the denervation atrophy and myofibrillar protein content.

10. the preservation of microorganism

In on March 26th, 1991 at The Agricultural Research Culture Collection(NRRL) (1815 North University Street, Peoria, Illi-nois, 61604) carried out following preservation.

Be loaded with plasmid pCMX-hCNTFR(I 2) intestinal bacteria, contain the expression plasmid of hCNTFR encoding sequence, given preserving number is NRRL B-18789.

The disclosed content of various reference, document that this paper quoted as proof all combines with the application also as a reference.

The present invention is not limited to the scope of disclosed embodiment among the structure of institute's preservation or the embodiment, and these all are for several aspect of the present invention is described, the suitable embodiment of any function all within the scope of the present invention.In addition, various changes shown here to the present invention and that describe all are conspicuous for the one of ordinary skilled in the art, and drop within the scope of appended claim.

Claims (36)

1, a kind of nucleic acid molecule of pure basically coding CNTF acceptor.

2, the nucleic acid molecule of claim 1, this molecule have the nucleotide sequence (SEQ ID No:1) as being drawn among Fig. 2 basically, or it is at least the fragment of 6 length of nucleotides.

3, the nucleic acid molecule of claim 2 is cDNA.

4, the pure substantially nucleic acid molecule of at least 6 nucleotide segments of the nucleotide sequence of being drawn among a kind of Fig. 2 of containing (SEQ ID No:1).

5, at least 6 nucleotide segments of the nucleotide sequence of being drawn among a kind of Fig. 2 of containing (SEQ ID No:1) but have the pure basically nucleic acid molecule of sudden change.

6, a kind of contain have the sequence (SEQ ID No:1) drawn as Fig. 2 basically but second kind of pure basically nucleic acid molecule of hybridization portion of first kind of nucleic acid molecule.

7, a kind of pure basically nucleic acid molecule, this molecule encoding have the protein of the aminoacid sequence of drawing as Fig. 2 basically (SEQ ID No:2), or its a part of or derivatives thereof of being made up of at least 6 amino acid.

8, a kind of biology that contains claim 1,2 or 7 nucleic acid molecule.

9, the biology of claim 8 is a kind of microorganisms.

10, the microorganism of claim 9 is a kind of bacteriums.

11, the microorganism of claim 9 is yeast.

12, the biology of claim 8 is a kind of transgenic animal.

13, a kind of cell that contains claim 1,2 or 7 nucleic acid molecule.

14, a kind of permanent clone that contains claim 1,2 or 7 nucleic acid molecule.

15, claim 1,2 or 7 nucleic acid molecule also contain the nucleotide sequence of can controlling gene expressing.

16, a kind of pure basically CNTF acceptor molecule, this molecule contain the aminoacid sequence (SEQ ID No:2) as drawing among Fig. 2 basically, or a part of being made up of at least 6 amino acid of tool.

17, a kind of monoclonal antibody that can discern the CNTFR acceptor of claim 16.

18, a kind of evaluation and the method for CNTF bonded cell comprises the receptor expression by this raji cell assay Raji CNTF.

19, the method for claim 18 comprises that also this probe contains at least 6 nucleotide segments of the sequence of drawing among Fig. 2 (SEQ ID No:1) by hybridizing to the existence of measuring CNTF acceptor-coding RNA on the nucleic acid probe from the sample of the suspension cell that contains CNTF acceptor coding RNA.

20, the method for claim 18 also comprises by (ⅰ) and can take place under the immunological characteristic bonded condition this cellular exposure in anti-CNTF receptor antibody, and (ⅱ) measures the existence that antibody and combining of this cell are measured the CNTF receptor protein then.

21, a kind of screening has the detection system of the active medicinal compound of CNTF, CNTF wherein comprises the cell that contains recombinant nucleic acid molecule, and this nucleic acid molecule contains basically as the nucleotide sequence of drawing among Fig. 2 (SEQ ID No:1) or its fragment of 6 length of nucleotides at least.

22, a kind of screening has the detection system of the active medicinal compound of CNTF, and CNTF wherein contains the cell of expression recombinant CNTF receptor protein or its functional activity part.

23, the detection system of claim 22, recombinant chou CNTF receptor protein wherein contain basically as the nucleotide sequence of drawing among Fig. 2 (SEQ ID No:2) or its fragment of 6 length of nucleotides at least.

24, the physiological experimental model system of a kind of research CNTF, CNTF wherein comprises the cell of the recombinant nucleic acid that contains coding CNTF acceptor, and compare the CNTF acceptor number that this cell increases at its surface expression with the cell of the recombinant nucleic acid that does not contain coding CNTF acceptor of same type.

25, the experimental model system of claim 24 is a kind of transgenic animal.

26, a kind of genetically modified non-human body transgenic animal that are loaded with the irrelevant recombinant chou CNTF acceptor of coding and cell surface.

27, a kind of being loaded with is coded in the genetically modified non-human body transgenic animal of transduction to adiaphorous recombinant chou CNTF acceptor in the CNTF biological response.

28, a kind of medicinal compositions contains CNTF receptor protein, peptide fragment, or derivatives thereof; And pharmaceutically useful carrier.

29, a kind of medicinal compositions contains protein and a kind of pharmaceutically useful carrier with aminoacid sequence, and aminoacid sequence wherein contains basically aminoacid sequence (SEQ ID No:2) or its 6 amino acid moieties as Fig. 2 drew.

30, a kind of evaluation about the method for the CNTF acceptor molecule that comprises IL-6 acceptor molecule family member comprises:

(ⅰ) screening is used for hybridizing to CNTF acceptor one coding nucleic acid clone's partly dna library;

(ⅱ) separation and breeding hybridize to the clone in the CNTF acceptor coding nucleic acid;

(ⅲ) be determined at and separate in the step (ⅱ) and the clone's of breeding nucleotide sequence; And

(ⅳ) nucleotide sequence that step (ⅲ) is measured is compared with known member's nucleotide sequence in the family.

31, a kind of pure basically molecule except that CNTF, this molecule and CNTF contention are combined on the CNTF acceptor.

32, a kind of auto-immune disease model system that contains the animal of using the CNTF acceptor immunity that causes the immunity amount.

33, a kind of second kind of nucleic acid molecule of pure basically additional claim 1 nucleic acid molecule.

34, a kind of pure basically additional claim 2 nucleic acid molecule or segmental second kind of nucleic acid molecule.

35, the cell composition of a kind of expression recombinant CNTF receptor protein of purifying basically or its functional activity part.

36, a kind of nucleic acid that contains the CNTF receptor nucleic acids sequence that is included among the plasmid pCMX-hCNTFR, it has been deposited in NRRL, and the preserving number that has is B-18789.

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